Antibody molecule for human GM-CSF receptor alpha

ABSTRACT

Binding members for alpha chain of receptor for granulocyte macrophage colony stimulating factor (GM-CSFRα), especially antibody molecules. Use of the binding members in treating inflammatory and autoimmune diseases, e.g. rheumatoid arthritis, asthma, allergic response, multiple sclerosis, myeloid leukaemia and atherosclerosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/GB2007/001108, filed Mar. 27, 2007, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 60/786,569, filed Mar. 27, 2006. Each of thereferenced applications is incorporated herein in its entirety.

The present invention relates to binding members for the alpha chain ofGranulocyte/Macrophage Colony Stimulating Factor Receptor (GM-CSFRα),especially anti-GMCSFRα antibody molecules. It also relates to use ofthese binding members in treating inflammatory, respiratory andautoimmune diseases mediated through GMCSFRα, including rheumatoidarthritis, chronic obstructive pulmonary disease and multiple sclerosis.

GM-CSF is a type I proinflammatory cytokine which enhances survival,proliferation and/or differentiation of a broad range of haematopoieticcell types including neutrophils, eosinophils, macrophages and theirprogenitor cells. The GM-CSF receptor is a member of the haematopoietinreceptor superfamily. It is heterodimeric, consisting of an alpha and abeta subunit. The alpha subunit is highly specific for GM-CSF whereasthe beta subunit is shared with other cytokine receptors, including IL3and IL5. This is reflected in a broader tissue distribution of the betareceptor subunit. The alpha subunit, GM-CSFRα, is primarily expressed onmyeloid cells and non-haematopoietic cells, such as neutrophils,macrophages, eosinophils, dendritic cells, endothelial cells andrespiratory epithelial cells. Full length GM-CSFRα is a 400 amino acidtype I membrane glycoprotein that belongs to the type I cytokinereceptor family, and consists of a 22 amino acid signal peptide(positions 1-22), a 298 amino acid extracellular domain (positions23-320), a transmembrane domain from positions 321-345 and a short 55amino acid intra-cellular domain. The signal peptide is cleaved toprovide the mature form of GM-CSFRα as a 378 amino acid protein. cDNAclones of the human and murine GM-CSFRα are available and, at theprotein level, the receptor subunits have 36% identity. GM-CSF is ableto bind with relatively low affinity to the α subunit alone (Kd 1-5 nM)but not at all to the β subunit alone. However, the presence of both αand β subunits results in a high affinity ligand-receptor complex(Kd≈100 pM). GM-CSF signalling occurs through its initial binding to theGM-CSFR α chain and then cross-linking with a larger subunit the commonβ chain to generate the high affinity interaction, which phosphorylatesthe JAK-STAT pathway. GM- CSFR binding to GMCSF is reviewed in ref. [1].This interaction is also capable of signalling through tyrosinephosphorylation and activation of the MAP kinase pathway.

Pathologically, GM-CSF has been shown to play a role in exacerbatinginflammatory, respiratory and autoimmune diseases. Neutralisation ofGM-CSF binding to GM-CSFRα is therefore a therapeutic approach totreating diseases and conditions mediated through GM-CSFR.

Nicola et al. [2] described a murine antibody against human GM-CSFRα,designated 2B7-17-A or “2B7”, which was reported to have a relativelyhigh affinity for human GM-CSFRα and to be a potent inhibitor of humanGM-CSF biological action in several different bioassays. Antibody 2B7 isavailable commercially from Chemicon as MAB1037, and the Product DataSheet for MAB1037 notes it is a potent inhibitor of GM-CSF biologicalaction. 2B7 was also disclosed in WO94/09149.

By using a combination of selections on naïve scFv phage libraries,random mutagenesis and appropriately designed biochemical and biologicalassays (see the Experimental Part below), we have identified highlypotent antibody molecules that bind to human GM-CSFRα and inhibit theaction of human GM-CSF at its receptor. The results presented hereinindicate that our antibodies bind a different region or epitope ofGM-CSFRα compared with the known anti-GM-CSFRα antibody 2B7, andsurprisingly are even more potent than 2B7 as demonstrated in a varietyof biological assays.

Accordingly, this invention relates to binding members that bind humanGM-CSFRα and inhibit binding of human GM-CSF to GM-CSFRα. Bindingmembers of the invention may be antagonists of GM-CSFR. The bindingmembers may be competitive reversible inhibitors of GM-CSF signallingthrough GM-CSFR.

Antibodies and other binding members of the invention are of particularvalue in binding and neutralising GM-CSFRα, and thus are of use intreatments for diseases mediated by GM-CSFRα, including inflammatory andautoimmune diseases, as indicated by the experimentation containedherein and further supporting technical literature. For example, we havedemonstrated in cell-based assays that antibodies of the invention areable to inhibit release of cytokines (e.g. IL-6 and TNFα) induced bynative. GM-CSF binding to its receptor. As explained in more detailbelow, inhibiting GM-CSF activity by blocking binding to GM-CSFRα is atherapeutic approach to treating such diseases as rheumatoid arthritis(PA), asthma, smoke-induced airway inflammation, chronic obstructivepulmonary disease (COPD), allergic response, multiple sclerosis (MS),myeloid leukaemia and atherosclerosis.

Binding members according to the invention generally bind theextracellular domain of GM-CSFRα. Preferably, a binding member of theinvention binds at least one residue of Tyr-Leu-Asp-Phe-Gln (YLDFQ), SEQID NO: 201, at positions 226 to 230 of mature human GM-CSFRα (SEQ ID NO:206). The binding member may bind at least one residue in the YLDFQsequence of human GM-CSFRα, e.g. it may bind one, two, three or fourresidues of the YLDFQ sequence. Thus, the binding member may recogniseone or more residues within this sequence, and optionally it may alsobind additional flanking residues or structurally neighbouring residuesin the extra-cellular domain of GM-CSFRα.

Binding may be determined by any suitable method, for example apeptide-binding scan may be used, such as a PEPSCAN-based enzyme linkedimmuno assay (ELISA), as described in detail elsewhere herein. In apeptide-binding scan, such as the kind provided by PEPSCAN Systems,short overlapping peptides derived from the antigen are systematicallyscreened for binding to a binding member. The peptides may be covalentlycoupled to a support surface to form an array of peptides. Briefly, apeptide binding scan (e.g. “PEPSCAN”) involves identifying (e.g. usingELISA) a set of peptides to which the binding member binds, wherein thepeptides have amino acid sequences corresponding to fragments of SEQ IDNO: 206 (e.g. peptides of about 15 contiguous residues of SEQ ID NO:206), and aligning the peptides in order to determine a footprint ofresidues bound by the binding member, where the footprint comprisesresidues common to overlapping peptides. In accordance with theinvention, the footprint identified by the peptide-binding scan orPEPSCAN may comprise at least one residue of YLDFQ corresponding topositions 226 to 230 of SEQ ID NO: 206. The footprint may comprise one,two, three, four or all residues of YLDFQ. A binding member according tothe invention may bind a peptide fragment (e.g. of 15 residues) of SEQID NO: 206 comprising one or more, preferably all, of residues YLDFQcorresponding to positions 226 to 230 of SEQ ID NO: 206, e.g. asdetermined by a peptide-binding scan or PEPSCAN method described herein.Thus, a binding member of the invention may bind a peptide having anamino acid sequence of 15 contiguous residues of SEQ ID NO: 206, whereinthe 15 residue sequence comprises at least one residue of, or at leastpartially overlaps with, YLDFQ at positions 226 to 230 of SEQ ID NO:206. Details of a suitable peptide-binding scan method for determiningbinding are set out in detail elsewhere herein. Other methods which arewell known in the art and could be used to determine the residues boundby an antibody, and/or to confirm peptide-binding scan (e.g. PEPSCAN)results, include site directed mutagenesis, hydrogen deuterium exchange,mass spectrometry, NMR, and X-ray crystallography.

Accordingly, a binding member of the invention preferably neutralisesGM-CSFRα. Neutralisation means reduction or inhibition of biologicalactivity of GM-CSFRα, e.g. reduction or inhibition of GM-CSF binding toGM-CSFRα, or of signalling by GM-CSFRα e.g. as measured byGM-CSFRα-mediated responses. The reduction or inhibition in biologicalactivity may be partial or total. The degree to which an antibodyneutralises GM-CSFRα is referred to as its neutralising potency. Potencymay be determined or measured using one or more assays known to theskilled person and/or as described or referred to herein. For example,the binding member may have neutralising activity in one or more of thefollowing assays:

-   -   Biochemical ligand binding assay    -   TF-1 proliferation assay    -   Human granulocyte shape change assay    -   Cynomolgus non human primate granulocyte shape change assay    -   Monocyte TNFα release assay    -   Granulocyte survival assay    -   Colony formation assay (inhibition of in vitro GM-CSF mediated        differentiation of blood cell progenitors)    -   Inhibition of GM-CSF bioactivity in vivo e.g. in chimaeric mice        with transgenic bone marrow expressing human GM-CSFR    -   Peripheral blood mononuclear cell cytokine release assay

Potency is normally expressed as an IC50 value, in pM unless otherwisestated. In functional assays, IC50 is the concentration that reduces abiological response by 50% of its maximum. In ligand-binding studies,IC50 is the concentration that reduces receptor binding by 50% ofmaximal specific binding level. IC50 may be calculated by plotting %maximal biological response (represented e.g. by cell proliferation,which may be measured as 3H thymidine incorporation in cpm, in aproliferation assay, by shape change in a shape change assay, by TNFαrelease in a TNFα release assay, by survival in a survival assay, bynumber of colonies in a colony formation assay, or by increase in spleenweight or decrease in circulating monocytes in chimaeric mice withtransgenic bone marrow expressing human GM-CSFR in a bioactivity test)or % specific receptor binding as a function of the log of the bindingmember concentration, and using a software program such as Prism(GraphPad) to fit a sigmoidal function to the data to generate IC50values.

An IC50 value may represent the mean of a plurality of measurements.Thus, for example, IC50 values may be obtained from the results oftriplicate experiments, and a mean IC50 value can then be calculated.

In the TF-1 proliferation assay, binding members of the inventionnormally have an IC50 of less than 1500 pM. For example, the IC50 may be<300, <60, <10, or <1.5 pM e.g. about 1.0 pM. Normally IC50 is at least0.5 or 1.0 nM. The known murine antibody 2B7 had an IC50 of about 1600pM in this assay. The TF-1 proliferation assay used herein was with afinal concentration of 7 pM human GM-CSF. Thus, IC50 neutralisingpotency in the TF-1 proliferation assay represents ability of a bindingmember to inhibit proliferation of TF-1 cells induced by 7 pM humanGM-CSF. For more details see the Assay Methods and Materials section.

A binding member of the invention may have a pA₂ more negative than −6,−7, −8, −9, −10, −10.5 or −11 in the TF-1 proliferation assay. Forexample, pA₂ may be about −10.5 or −11. Calculation and significance ofpA₂ values is discussed in detail in the Experimental Part under AssayMethods and Materials.

In the human granulocyte shape change assay, binding members of theinvention normally have an IC50 of less than 100 pM, e.g. less than 50pM or less than 30, 25, 20, 15 or 10 pM. Normally IC50 is at least 5, 6or 7 pM. The known murine antibody 2B7 in contrast is less potent with ameasured IC50 of 477 pM in this assay. The human granulocyte shapechange assay used herein was with a final concentration of 7 pM humanGM-CSF. Thus, IC50 neutralising potency in the human granulocyte shapechange assay represents ability of a binding member to inhibit shapechange of human granulocytes induced by 7 pM human GM-CSF. For moredetails see the Assay Methods and Materials section.

In the cynomolgus granulocyte shape change assay, binding members of theinvention normally have an IC50 of less than 20 pM, typically less than10, 5 or 2.5 pM. IC50 may be at least 0.5, 1 or 1.5 pM. The known murineantibody 2B7 had an IC50 of 26 pM when tested in this assay. Thecynomolgus granulocyte shape change assay used herein was with a finalconcentration of 7 pM human GM-CSF. Thus, IC50 neutralising potency inthe cynomolgus granulocyte shape change assay represents ability of abinding member to inhibit shape change of cynomolgus granulocytesinduced by 7 human pM GM-CSF. For more details see the Assay Methods andMaterials section.

A binding member of the invention may have a pA₂ more negative than −6,−7, −8, −9, −10, −10.5 or −11 in the human and/or cynomologus shapechange assay. Preferably the pA₂ is about −10 or −11.

In the monocyte TNFα release assay, binding members of the inventionnormally have an IC50 of less than 150 pM, typically less than 110 pMe.g. less than 100 pM. IC50 may be at least 30 or 40 pM. The monocyteTNFα release assay used herein was with a final concentration of 1 nMhuman GM-CSF. Thus, IC50 neutralising potency in the monocyte TNFαrelease assay represents ability of a binding member to inhibit TNFαrelease from human monocytes stimulated with 1 nM human GM-CSF. For moredetails see the Assay Methods and Materials section.

In the granulocyte survival assay, binding members of the inventionnormally have an IC50 of less than 1000 pM, typically less than 850 pM.IC50 may be less than 500, 250, 150, 100, 50, 30, 20 or 10 pM. IC50 maybe at least 5 pM. The known murine antibody 2B7 is inactive in thisassay up to a concentration of 83 nM. The granulocyte survival assayused herein was with a final concentration of 7 pM human GM-CSF. Thus,IC50 neutralising potency in the granulocyte survival assay representsability of a binding member to inhibit survival of human granulocytesinduced by 7 pM human GM-CSF. For more details see the Assay Methods andMaterials section.

In the colony formation assay, binding members of the invention may havean IC50 of less than 5, less than 2.5, less than 1 or less than 0.3μg/ml. Preferably the IC50 is 0.25 μg/ml or less, e.g. less than 0.1μg/ml. IC50 may be at least 0.05 μg/ml. The known murine antibody 2B7has little if any activity in this assay up to a concentration of 10μg/ml (67 nM). The colony formation assay used herein was with a finalconcentration of 10 ng/ml human GM-CSF. Thus, IC50 neutralising potencyin the colony formation assay represents ability of a binding member toinhibit colony formation induced by 10 ng/ml human GM-CSF. For moredetails see the Assay Methods and Materials section.

A binding member of the invention may show a dose dependent ability toinhibit increase in spleen weight and/or to inhibit a GM-CSF induceddecrease in circulating monocytes in chimaeric mice with transgenic bonemarrow expressing human GM-CSFR, that are treated with human GM-CSF.IC50 for inhibition of increased spleen weight may be less than 5, lessthan 2.5, less than 2, less than 1 or less than 0.75 mg/kg. IC50 may beat least 0.5 mg/kg in some embodiments.

Additionally, binding kinetics and affinity of binding members for humanGM-CSFRα may be determined, for example by surface plasmon resonancee.g. using BIACORE®. Binding members of the invention normally have a KDof less than 5 nM and more preferably less than 4, 3, 2 or 1 nM.Preferably, KD is less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or0.15 nM.

Binding members of the invention normally bind non-human primateGM-CSFRα e.g. cynomolgus GM-CSFRα in addition to human GM-CSFRdα. Asthere is a low homology between human and murine GM-CSF receptor(approximately 36%), binding members of the invention will generally notbind or cross-react with the murine receptor.

Normally a binding member of the invention comprises an antibodymolecule, e.g. a whole antibody or antibody fragment, as discussed inmore detail below. Preferably, an antibody molecule of the invention isa human antibody molecule.

A binding member of the invention normally comprises an antibody VHand/or VL domain. VH domains and VL domains of binding members are alsoprovided as part of the invention. Within each of the VH and VL domainsare complementarity determining regions (“CDRs”), and framework regions,(“FRs”). A VH domain comprises a set of HCDRs and a VL domain comprisesa set of LCDRs. An antibody molecule typically comprises an antibody VHdomain comprising a VH CDR1, CDR2 and CDR3 and a framework. It mayalternatively or also comprise an antibody VL domain comprising a VLCDR1, CDR2 and CDR3 and a framework. A VH or VL domain frameworkcomprises four framework regions, FR1, FR2, FR3 and FR4, interspersedwith CDRs in the following structure:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Examples of antibody VH and VL domains, FRs and CDRs according to thepresent invention are as listed in the appended sequence listing thatforms part of the present disclosure. All VH and VL sequences, CDRsequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosedherein represent aspects and embodiments of the invention. Thus, anaspect of the invention is a VH domain of a binding member according tothe invention. A “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, aset of HCDRs means HCDR1, HCDR2 and HCDR3, and a set of LCDRs meansLCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs”includes HCDRs and LCDRs. Typically binding members of the invention aremonoclonal antibodies (mAb).

As described in more detail in the Experimental Part, we identified apanel of antibody molecules that bind GM-CSFRα. We also identifiedcertain residues within the complementarity determining regions (CDRs)of the VH and VL domains that are especially important for receptorbinding and neutralisation potency. Since the CDRs are primarilyresponsible for determining binding and specificity of a binding member,one or more CDRs having the appropriate residues as defined herein maybe used and incorporated into any suitable framework, for example anantibody VH and/or VL domain framework, or a non-antibody proteinscaffold, as described in more detail elsewhere herein. For example, oneor more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule ordifferent antibody molecules. For example, an antibody molecule maycomprise CDRs as disclosed herein and framework regions of humangermline gene segment sequences. An antibody may be provided with a setof CDRs within a framework which may be subject to germlining, where oneor more residues within the framework are changed to match the residuesat the equivalent position in the most similar human germline framework.Thus, antibody framework regions are preferably germline and/or human.

We carried out an investigation into which residues of a candidateantibody were important for antigen recognition, following the methodset out in the experimental section, and then performed sequenceanalysis of 160 clones showing a potency at least 5-fold higher than theparent antibody clone in a biological assay. The results indicated thefollowing positions as contributing to antigen binding: Kabat residues27A, 27B, 27C, 32, 51, 52, 53, 90, 92 and 96 in the VL domain and Kabatresidues 17, 34, 54, 57, 95, 97, 99 and 100B in the VH domain. Inpreferred embodiments of the invention, one or more of these Kabatresidues is the Kabat residue present at that position for one or moreof the antibody clones numbered 1, 2 and 4-20 whose sequences aredisclosed in the appended sequence listing. In various embodiments theresidue may be the same as, or may differ from, the residue present atthat position in antibody 3.

Our analysis indicated 4 residue positions in the CDRs that have aparticularly strong influence on receptor binding: H97, H100B, L90 andL92 (Kabat numbering). Preferably, H97 of VH CDR3 is S. The serineresidue at this position was observed in all 160 clones and thereforerepresents an important residue for antigen recognition.

Preferably, a VH CDR3 comprises one or more of the following residues:

-   V, N, A or L at Kabat residue H95, most preferably V;-   S, F, H, P, T or W at Kabat residue H99, most preferably S;-   A, T, P, S, V or H at Kabat residue H100B, most preferably A or T.

Preferably, Kabat residue H34 in VH CDR1 is I. Preferably, VH CDR2comprises E at Kabat residue H54 and/or I at Kabat residue H57.

Where the binding member comprises an antibody VH domain, Kabat residueH17 in the VH domain framework is preferably S. Kabat residue H94 ispreferably I or a conservative substitution thereof (e.g. L, V, A or M).Normally H94 is I.

Preferably, a VL CDR3 comprises one or more of the following residues:

-   S, T or M at Kabat residue L90, most preferably S or T;-   D, E, Q, S, M or T at Kabat residue L92, most preferably D or E;-   A, P, S, T, I, L, M or V at Kabat residue L96, most preferably S, P,    I or V, especially S.

Kabat residue L95A in VL CDR3 is preferably S.

Preferably, a VL CDR1 comprises one or more of the following residues:

-   S at Kabat residue 27A;-   N at Kabat residue 27B;-   I at Kabat residue 27C;-   D at Kabat residue 32.

Preferably, a VL CDR2 comprises one or more of the following residues:

-   N at Kabat residue 51;-   N at Kabat residue 52;-   K at Kabat residue 53.

In a preferred embodiment, a binding member of the invention comprisesone or more CDRs selected from the VH and VL CDRs, i.e. a VH CDR1, 2and/or 3 and/or a VL CDR 1, 2 and/or 3 of any of antibodies 1, 2 or 4 to20 as shown in the sequence listing, or of the parent antibody 3. In apreferred embodiment a binding member of the invention comprises a VHCDR3 of any of the following antibody molecules: Antibody 1 (SEQ ID NO5); Antibody 2 (SEQ ID NO 15); Antibody 3 (SEQ ID NO 25); Antibody 4(SEQ ID NO 35); Antibody 5 (SEQ ID NO 45); Antibody 6 (SEQ ID NO 55);Antibody 7 (SEQ ID NO 65); Antibody 8 (SEQ ID NO 75); Antibody 9 (SEQ IDNO 85); Antibody 10 (SEQ ID NO 95); Antibody 11 (SEQ ID NO 105);Antibody 12 (SEQ ID NO 115); Antibody 13 (SEQ ID NO 125); Antibody 14(SEQ ID NO 135); Antibody 15 (SEQ ID NO 145); Antibody 16 (SEQ ID NO155); Antibody 17 (SEQ ID NO 165); Antibody 18 (SEQ ID NO 175); Antibody19 (SEQ ID NO 185); Antibody 20 (SEQ ID NO 195). Preferably, the bindingmember additionally comprises a VH CDR1 of SEQ ID NO: 3 or SEQ ID NO:173 and/or a VH CDR2 of SEQ ID NO: 4. Preferably, a binding membercomprising VH CDR3 of SEQ ID NOS: 175 comprises a VH CDR1 of SEQ ID NO:173, but may alternatively comprise a VH CDR1 of SEQ ID NO: 3.

Preferably the binding member comprises a set of VH CDRs of one of thefollowing antibodies: Antibody 1 (SEQ ID NOS: 3-5); Antibody 2 ( SEQ IDNOS: 3, 4, 15); Antibody 3 (SEQ ID NOS: 3, 4, 25); Antibody 4 (SEQ IDNOS: 3, 4, 35); Antibody 5 (SEQ ID NOS: 3, 4, 45); Antibody 6 (SEQ IDNOS: 3, 4, 55); Antibody 7 (SEQ ID NOS: 3, 4, 65); Antibody 8 (SEQ IDNOS: 3, 4,75); Antibody 9 ( SEQ ID NOS: 3, 4, 85); Antibody 10 (SEQ IDNOS: 3, 4, 95); Antibody 11 (SEQ ID NOS: 3, 4, 105); Antibody 12 (SEQ IDNOS: 3, 4, 115); Antibody 13 (SEQ ID NOS: 3, 4, 125); Antibody 14 (SEQID NOS: 3,4 135); Antibody 15 (SEQ ID NOS: 3, 4, 15); Antibody 16 (SEQID NOS: 3, 4, 155); Antibody 17 (SEQ ID NOS: 3, 4, 165); Antibody 18(SEQ ID NOS: 173,4, 175); Antibody 19 (SEQ ID NOS: 3, 4, 185); Antibody20 (SEQ ID NOS: 3, 4, 195). Optionally it may also comprise a set of VLCDRs of one of these antibodies, and the VL CDRs may be from the same ora different antibody as the VH CDRs. Generally, a VH domain is pairedwith a VL domain to provide an antibody antigen-binding site, althoughin some embodiments a VH or VL domain alone may be used to bind antigen.Light-chain promiscuity is well established in the art, and thus the VHand VL domain need not be from the same clone as disclosed herein.

A binding member may comprise a set of H and/or L CDRs of any ofantibodies 1 to 20 with one or more substitutions, for example ten orfewer, e.g. one, two, three, four or five, substitutions within thedisclosed set of H and/or L CDRs. Preferred substitutions are at Kabatresidues other than Kabat residues 27A, 27B, 27C, 32, 51, 52, 53, 90, 92and 96 in the VL domain and Kabat residues 34, 54, 57, 95, 97, 99 and100B in the VH domain. Where substitutions are made at these positions,the substitution is preferably for a residue indicated herein as being apreferred residue at that position.

In a preferred embodiment, a binding member of the invention is anisolated human antibody molecule having a VH domain comprising a set ofHCDRs in a human germline framework, e.g. human germline framework fromthe heavy chain VH1 or VH3 family. In a preferred embodiment, theisolated human antibody molecule has a VH domain comprising a set ofHCDRs in a human germline framework VH1 DP5 or VH3 DP47. Thus, the VHdomain framework regions may comprise framework regions of humangermline gene segment VH1 DP5 or VH3 DP47. The amino acid sequence of VHFR1 may be SEQ ID NO: 251. The amino acid sequence of VH FR2 may be SEQID NO: 252. The amino acid sequence of VH FR3 may be SEQ ID NO: 253. Theamino acid sequence of VH FR4 may be SEQ ID NO: 254.

Normally the binding member also has a VL domain comprising a set ofLCDRs, preferably in a human germline framework e.g. a human germlineframework from the light chain Vlambda 1 or Vlambda 6 family. In apreferred embodiment, the isolated human antibody molecule has a VLdomain comprising a set of LCDRs in a human germline framework VLambda 1DPL8 or VLambda 1 DPL3 or VLambda 6_(—)6a. Thus, the VL domain frameworkmay comprise framework regions of human germline gene segment VLambda 1DPL8, VLambda 1 DPL3 or VLambda 6_(—)6a. The VL domain FR4 may comprisea framework region of human germline gene segment JL2. The amino acidsequence of VL FR1 may be SEQ ID NO: 255. The amino acid sequence of VLFR2 may be SEQ ID NO: 256. The amino acid sequence of VL FR3 may be 257.The amino acid sequence of VL FR4 may be SEQ ID NO: 258.

A non-germlined antibody has the same CDRs, but different frameworks,compared with a germlined antibody.

A binding member of the invention may compete for binding to GM-CSFRαwith any binding member disclosed herein e.g. antibody 3 or any ofantibodies 1, 2 or 4-20. Thus a binding member may compete for bindingto GM-CSFRα with an antibody molecule comprising the VH domain and VLdomain of any of antibodies 1, 2 or 4-20. Competition between bindingmembers may be assayed easily in vitro, for example by tagging areporter molecule to one binding member which can be detected in thepresence of one or more other untagged binding members, to enableidentification of binding members which bind the same epitope or anoverlapping epitope.

Competition may be determined for example using ELISA in which e.g. theextracellular domain of GM-CSFRα, or a peptide of the extracellulardomain, is immobilised to a plate and a first tagged binding memberalong with one or more other untagged binding members is added to theplate. Presence of an untagged binding member that competes with thetagged binding member is observed by a decrease in the signal emitted bythe tagged binding member. Similarly, a surface plasmon resonance assaymay be used to determine competition between binding members.

In testing for competition a peptide fragment of the antigen may beemployed, especially a peptide including or consisting essentially of anepitope or binding region of interest. A peptide having the epitope ortarget sequence plus one or more amino acids at either end may be used.Binding members according to the present invention may be such thattheir binding for antigen is inhibited by a peptide with or includingthe sequence given.

Binding members that bind a peptide may be isolated for example from aphage display library by panning with the peptide(s).

The present invention also provides the use of a binding member as abovefor measuring antigen levels in a competition assay, that is to say amethod of measuring the level of antigen in a sample by employing abinding member as provided by the present invention in a competitionassay. This may be where the physical separation of bound from unboundantigen is not required. Linking a reporter molecule to the bindingmember so that a physical or optical change occurs on binding is onepossibility. The reporter molecule may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

The present invention also provides for measuring levels of antigendirectly, by employing a binding member according to the invention forexample in a biosensor system.

The present invention provides a method comprising causing or allowingbinding of a binding member as provided herein to GM-CSFRα. Such bindingmay take place in vivo, e.g. following administration of a bindingmember, or nucleic acid encoding a binding member, or it may take placein vitro, for example in ELISA, Western blotting, immunocytochemistry,immuno-precipitation, affinity chromatography, or cell based assays suchas a TF-1 assay.

The amount of binding of binding member to GM-CSFRα may be determined.Quantitation may be related to the amount of the antigen in a testsample, which may be of diagnostic or prognostic interest.

A kit comprising a binding member or antibody molecule according to anyaspect or embodiment of the present invention is also provided as anaspect of the present invention. In a kit of the invention, the bindingmember or antibody molecule may be labelled to allow its reactivity in asample to be determined, e.g. as described further below. Components ofa kit are generally sterile and in sealed vials or other containers.Kits may be employed in diagnostic analysis or other methods for whichantibody molecules are useful. A kit may contain instructions for use ofthe components in a method, e.g. a method in accordance with the presentinvention. Ancillary materials to assist in or to enable performing sucha method may be included within a kit of the invention.

The reactivities of antibodies in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the antibody. Bound antigen is physicallyseparated from unbound antigen and the amount of radioactive antigenbound to the antibody determined. The more antigen there is in the testsample the less radioactive antigen will bind to the antibody. Acompetitive binding assay may also be used with non-radioactive antigen,using antigen or an analogue linked to a reporter molecule. The reportermolecule may be a fluorochrome, phosphor or laser dye with spectrallyisolated absorption or emission characteristics. Suitable fluorochromesinclude fluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine. Other reporters includemacromolecular colloidal particles or particulate material such as latexbeads that are coloured, magnetic or paramagnetic, and biologically orchemically active agents that can directly or indirectly causedetectable signals to be visually observed, electronically detected orotherwise recorded. These molecules may be enzymes, which catalysereactions that develop, or change colours or cause changes in electricalproperties, for example. They may be molecularly excitable, such thatelectronic transitions between energy states result in characteristicspectral absorptions or emissions. They may include chemical entitiesused in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in samples (normal and test).

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a binding member, VH domain and/orVL domain according to the present invention. Nucleic acid may includeDNA and/or RNA, and may be wholly or partially synthetic. Reference to anucleotide sequence as set out herein encompasses a DNA molecule withthe specified sequence, and encompasses a RNA molecule with thespecified sequence in which U is substituted for T, unless contextrequires otherwise. In a preferred aspect, the present inventionprovides a nucleic acid that codes for a CDR or set of CDRs or VH domainor VL domain or antibody antigen-binding site or antibody molecule, e.g.scFv or IgG1 or IgG4, of the invention as defined herein. The presentinvention also provides constructs in the form of plasmids, vectors,transcription or expression cassettes which comprise at least onepolynucleotide as above.

A further aspect is a host cell transformed with or containing nucleicacid of the invention. Such a host cell may be in vitro and may be inculture. Such a host cell may be in vivo. In vivo presence of the hostcell may allow intracellular expression of the binding members of thepresent invention as “intrabodies” or intracellular antibodies.Intrabodies may be used for gene therapy.

A still further aspect provides a method comprising introducing suchnucleic acid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay be incorporated into the host cell or into an artificial chromosome.Incorporation may be either by random or targeted integration of one ormore copies at single or multiple loci. For bacterial cells, suitabletechniques may include calcium chloride transformation, electroporationand transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences that promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method that comprises using aconstruct as stated above in an expression system in order to express abinding member or polypeptide as above. Thus, methods of preparing abinding member, a VH domain and/or a VL domain of the invention, arefurther aspects of the invention. A method may comprise expressing saidnucleic acid under conditions to bring about production of said bindingmember, VH domain and/or VL domain, and recovering it. Such a method maycomprise culturing host cells under conditions for production of saidbinding member or antibody domain.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, yeast and baculovirus systemsand transgenic plants and animals. The expression of antibodies andantibody fragments in prokaryotic cells is well established in the art[3]. A common, preferred bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a binding member[4,5,6]. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 ratmyeloma cells, human embryonic kidney cells, human embryonic retinacells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. 'phage, as appropriate [7]. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Ausubel et al. [8].

The present invention provides a method of obtaining one or more bindingmembers able to bind the antigen, the method including bringing intocontact a library of binding members according to the invention and saidantigen, and selecting one or more binding members of the library ableto bind said antigen.

The library may be displayed on particles or molecular complexes, e.g.replicable genetic packages such as yeast, bacterial or bacteriophage(e.g. T7) particles, or covalent, ribosomal or other in vitro displaysystems, each particle or molecular complex containing nucleic acidencoding the antibody VH variable domain displayed on it, and optionallyalso a displayed VL domain if present. Following selection of bindingmembers able to bind the antigen and displayed on bacteriophage or otherlibrary particles or molecular complexes, nucleic acid may be taken froma bacteriophage or other particle or molecular complex displaying a saidselected binding member. Such nucleic acid may be used in subsequentproduction of a binding member or an antibody VH or VL variable domainby expression from nucleic acid with the sequence of nucleic acid takenfrom a bacteriophage or other particle or molecular complex displaying asaid selected binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected binding member may beprovided in isolated form, as may a binding member comprising such a VHdomain.

An antibody VL variable domain with the amino acid sequence of anantibody VL variable domain of a said selected binding member may beprovided in isolated form, as may a binding member comprising such a VLdomain.

Ability to bind GM-CSFRα may be further tested, also ability to competewith any of antibodies 1 to 20 (e.g. in scFv format and/or IgG format,e.g. IgG1 or IgG4) for binding to GM-CSFRα. Ability to neutraliseGM-CSFRα may be tested.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein can beobtained by means of methods of sequence alteration or mutation andscreening, and can be employed in binding members for GM-CSFRα.Following the lead of computational chemistry in applying multivariatedata analysis techniques to the structure/property-activityrelationships [9] quantitative activity-property relationships ofantibodies can be derived using well-known mathematical techniques suchas statistical regression, pattern recognition and classification [10,11, 12, 13, 14, 15]. The properties of antibodies can be derived fromempirical and theoretical models (for example, analysis of likelycontact residues or calculated physicochemical property) of antibodysequence, functional and three-dimensional structures and theseproperties can be considered singly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis formed by six loops of polypeptide: three from the light chainvariable domain (VL) and three from the heavy chain variable domain(VH). Analysis of antibodies of known atomic structure has elucidatedrelationships between the sequence and three-dimensional structure ofantibody combining sites [16,17]. These relationships imply that, exceptfor the third region (loop) in VH domains, binding site loops have oneof a small number of main-chain conformations: canonical structures. Thecanonical structure formed in a particular loop has been shown to bedetermined by its size and the presence of certain residues at key sitesin both the loop and in framework regions [16,17].

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain binding.These predictions can be backed up by comparison of the predictions tothe output from lead optimization experiments. In a structural approach,a model can be created of the antibody molecule [18] using any freelyavailable or commercial package such as WAM [19]. A proteinvisualisation and analysis software package such as Insight II(Accelrys, Inc.) or Deep View [20] may then be used to evaluate possiblesubstitutions at each position in the CDR. This information may then beused to make substitutions likely to have a minimal or beneficial effecton activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and binding membersgenerally are available in the art. Variant sequences may be made, withsubstitutions that may or may not be predicted to have a minimal orbeneficial effect on activity, and tested for ability to bind and/orneutralise GM-CSFRα and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, maybe 5, 4, 3, 2 or 1. Alterations may be made in one ormore framework regions and/or one or more CDRs.

Preferably alterations do not result in loss of function, so a bindingmember comprising a thus-altered amino acid sequence preferably retainsan ability to bind and/or neutralise GM-CSFRα. More preferably, itretains the same quantitative binding and/or neutralising ability as abinding member in which the alteration is not made, e.g. as measured inan assay described herein. Most preferably, the binding membercomprising a thus-altered amino acid sequence has an improved ability tobind or neutralise GM-CSFRα compared with a binding member in which thealteration is not made, e.g. as measured in an assay described herein.

Alteration may comprise replacing one or more amino acid residue with anon-naturally occurring or non-standard amino acid, modifying one ormore amino acid residue into a non-naturally occurring or non-standardform, or inserting one or more non-naturally occurring or non-standardamino acid into the sequence. Preferred numbers and locations ofalterations in sequences of the invention are described elsewhereherein. Naturally occurring amino acids include the 20 “standard”L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C,K, R, H, D, E by their standard single-letter codes. Non-standard aminoacids include any other residue that may be incorporated into apolypeptide backbone or result from modification of an existing aminoacid residue. Non-standard amino acids may be naturally occurring ornon-naturally occurring. Several naturally occurring non-standard aminoacids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine,3-methylhistidine, N-acetylserine, etc. [21]. Those amino acid residuesthat are derivatised at their N-alpha position will only be located atthe N-terminus of an amino-acid sequence. Normally in the presentinvention an amino acid is an L-amino acid, but in some embodiments itmay be a D-amino acid. Alteration may therefore comprise modifying anL-amino acid into, or replacing it with, a D-amino acid. Methylated,acetylated and/or phosphorylated forms of amino acids are also known,and amino acids in the present invention may be subject to suchmodification.

Amino acid sequences in antibody domains and binding members of theinvention may comprise non-natural or non-standard amino acids describedabove. In some embodiments non-standard amino acids (e.g. D-amino acids)may be incorporated into an amino acid sequence during synthesis, whilein other embodiments the non-standard amino acids may be introduced bymodification or replacement of the “original” standard amino acids aftersynthesis of the amino acid sequence.

Use of non-standard and/or non-naturally occurring amino acids increasesstructural and functional diversity, and can thus increase the potentialfor achieving desired GM-CSFRα binding and neutralising properties in abinding member of the invention. Additionally, D-amino acids andanalogues have been shown to have better pharmacokinetic profilescompared with standard L-amino acids, owing to in vivo degradation ofpolypeptides having L-amino acids after administration to an animal.

As noted above, a CDR amino acid sequence substantially as set outherein is preferably carried as a CDR in a human antibody variabledomain or a substantial portion thereof. The HCDR3 sequencessubstantially as set out herein represent preferred embodiments of thepresent invention and it is preferred that each of these is carried as aHCDR3 in a human heavy chain variable domain or a substantial portionthereof.

Variable domains employed in the invention may be obtained or derivedfrom any germline or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A CDR sequence of the invention (e.g.CDR3) may be introduced into a repertoire of variable domains lacking aCDR (e.g. CDR3), using recombinant DNA technology.

For example, Marks et al. (1992) [22] describe methods of producingrepertoires of antibody variable domains in which consensus primersdirected at or adjacent to the 5′ end of the variable domain area areused in conjunction with consensus primers to the third framework regionof human VH genes to provide a repertoire of VH variable domains lackinga CDR3. Marks et al. further describe how this repertoire may becombined with a CDR3 of a particular antibody. Using analogoustechniques, the CDR3-derived sequences of the present invention may beshuffled with repertoires of VH or VL domains lacking a CDR3, and theshuffled complete VH or VL domains combined with a cognate VL or VHdomain to provide binding members of the invention. The repertoire maythen be displayed in a suitable host system such as the phage displaysystem of WO92/01047 or any of a subsequent large body of literature,including ref. [23], so that suitable binding members may be selected. Arepertoire may consist of from anything from 10⁴ individual membersupwards, for example from 10⁶ to 10⁸ or 10¹⁰ members. Other suitablehost systems include yeast display, bacterial display, T7 display, viraldisplay, cell display, ribosome display and covalent display. Analogousshuffling or combinatorial techniques are also disclosed by Stemmer(1994)[24], who describes the technique in relation to a β-lactamasegene but observes that the approach may be used for the generation ofantibodies.

A further alternative is to generate novel VH or VL regions carryingCDR-derived sequences of the invention using random mutagenesis of oneor more selected VH and/or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al.(1992) [25], who used error-prone PCR. In preferred embodiments one ortwo amino acid substitutions are made within a set of HCDRs and/orLCDRs. Another method that may be used is to direct mutagenesis to CDRregions of VH or VL genes [26,27].

A further aspect of the invention provides a method for obtaining anantibody antigen-binding site for GM-CSFRα antigen, the methodcomprising providing by way of addition, deletion, substitution orinsertion of one or more amino acids in the amino acid sequence of a VHdomain set out herein a VH domain which is an amino acid sequencevariant of the VH domain, optionally combining the VH domain thusprovided with one or more VL domains, and testing the VH domain or VH/VLcombination or combinations to identify a binding member or an antibodyantigen-binding site for GM-CSFRα antigen and optionally with one ormore preferred properties, preferably ability to neutralise GM-CSFRαactivity. Said VL domain may have an amino acid sequence which issubstantially as set out herein.

An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

A further aspect of the invention provides a method of preparing abinding member for GM-CSFRα antigen, which method comprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain which either include a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a VH        CDR3 such that said donor nucleic acid is inserted into the CDR3        region in the repertoire, so as to provide a product repertoire        of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting a binding member for GM-CSFRα; and    -   (e) recovering said binding member or nucleic acid encoding it.

Again, an analogous method may be employed in which a VL CDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain that either include a CDR3 to be replaced or lack a CDR3 encodingregion.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a bindingmember or binding members for GM-CSFRα.

In a preferred embodiment, one or more HCDR1, HCDR2 and HCDR3, e.g. aset of HCDRs of Antibody 1 (SEQ ID NOS: 3-5); Antibody 2 (SEQ ID NOS: 3,4, 15); Antibody 4 (SEQ ID NOS: 3, 4, 35); Antibody 5 (SEQ ID NOS: 3, 4,45); Antibody 6 (SEQ ID NOS: 3, 4, 55); Antibody 7 (SEQ ID NOS: 3, 4,65); Antibody 8 (SEQ ID NOS: 3, 4, 75); Antibody 9 (SEQ ID NOS: 3, 4,85); Antibody 10 (SEQ ID NOS: 3, 4, 95); Antibody 11 (SEQ ID NOS: 3, 4,105); Antibody 12 (SEQ ID NOS: 3, 4, 115); Antibody 13 (SEQ ID NOS: 3,4, 125); Antibody 14 (SEQ ID NOS: 3, 4, 135); Antibody 15 (SEQ ID NOS:3, 4, 145); Antibody 16 (SEQ ID NOS: 3, 4, 155); Antibody 17 (SEQ IDNOS: 3, 4, 165); Antibody 18 (SEQ ID NOS: 173, 4, 175); Antibody 19 (SEQID NOS: 3, 4, 185) or Antibody 20 (SEQ ID NOS: 3, 4, 195); or optionallyAntibody 3 (SEQ ID NOS: 3, 4, 25), may be employed, and/or one or moreLCDR1, LCDR2 and LCDR3 e.g. a set of LCDRs of Antibody 1 (SEQ ID NOS:8-10); Antibody 2 (SEQ ID NOS: 8, 9, 20); Antibody 4 (SEQ ID NOS: 8, 9,20); Antibody 5 (SEQ ID NOS: 8, 9, 20); Antibody 6 (SEQ ID NOS: 8, 9,60); Antibody 7 (SEQ ID NOS: 8, 9, 20); Antibody 8 (SEQ ID NOS: 8, 9,20); Antibody 9 (SEQ ID NOS: 8, 9, 20); Antibody 10 (SEQ ID NOS: 8, 9,20); Antibody 11 (SEQ ID NOS: 8, 9, 20); Antibody 12 (SEQ ID NOS: 8, 9,120); Antibody 13 (SEQ ID NOS: 8, 9, 130); Antibody 14 (SEQ ID NOS: 8,9, 140); Antibody 15 (SEQ ID NOS: 8, 9, 150); Antibody 16 (SEQ ID NOS:8, 9, 160); Antibody 17 (SEQ ID NOS: 8, 9, 170); Antibody 18 (SEQ IDNOS: 8, 9, 180); Antibody 19 (SEQ ID NOS: 8, 9, 190) or Antibody 20 (SEQID NOS: 8, 9, 200); or optionally Antibody 3 (SEQ ID NOS: 8, 9, 20), maybe employed.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of binding members ofthe present invention made by recombinant DNA techniques may result inthe introduction of N- or C-terminal residues encoded by linkersintroduced to facilitate cloning or other manipulation steps. Othermanipulation steps include the introduction of linkers to join variabledomains of the invention to further protein sequences including antibodyconstant regions, other variable domains (for example in the productionof diabodies) or detectable/functional labels as discussed in moredetail elsewhere herein.

Although in a preferred aspect of the invention binding memberscomprising a pair of VH and VL domains are preferred, single bindingdomains based on either VH or VL domain sequences form further aspectsof the invention. It is known that single immunoglobulin domains,especially VH domains, are capable of binding target antigens. Forexample, see the discussion of dabs elsewhere herein.

In the case of either of the single binding domains, these domains maybe used to screen for complementary domains capable of forming atwo-domain binding member able to bind GM-CSFRα. This may be achieved byphage display screening methods using the so-called hierarchical dualcombinatorial approach as disclosed in WO92/01047, in which anindividual colony containing either an H or L chain clone is used toinfect a complete library of clones encoding the other chain (L or H)and the resulting two-chain binding member is selected in accordancewith phage display techniques such as those described in that referenceand [22].

Further aspects of the present invention provide for compositionscontaining binding members of the invention and at least one additionalcomponent, e.g. a composition comprising a binding member and apharmaceutically acceptable excipient. Such compositions may be used inmethods of inhibiting or neutralising GM-CSFRα, including methods oftreatment of the human or animal body by therapy.

The invention provides heterogeneous preparations comprisinganti-GM-CSFRα antibody molecules. For example, such preparations may bemixtures of antibodies with full-length heavy chains and heavy chainslacking the C-terminal lysine, with various degrees of glycosylationand/or with derivatized amino acids, such as cyclization of anN-terminal glutamic acid to form a pyroglutamic acid residue.

Aspects of the invention include methods of treatment comprisingadministration of a binding member as provided, pharmaceuticalcompositions comprising such a binding member, and use of such a bindingmember in the manufacture of a medicament, for example in a method ofmaking a medicament or pharmaceutical composition comprising formulatingthe binding member with a pharmaceutically acceptable excipient.

Anti-GM-CSFRα treatment may be given orally (for example nanobodies), byinjection (for example, subcutaneously, intravenously, intra-arterially,intra-articularly, intraperitoneal or intramuscularly), by inhalation,by the intravesicular route (instillation into the urinary bladder), ortopically (for example intraocular, intranasal, rectal, into wounds, onskin). The treatment may be administered by pulse infusion, particularlywith declining doses of the binding member. The route of administrationcan be determined by the physicochemical characteristics of thetreatment, by special considerations for the disease or by therequirement to optimise efficacy or to minimise side-effects. It isenvisaged that anti-GM-CSFRα treatment will not be restricted to use inthe clinic. Therefore, subcutaneous injection using a needle free deviceis also preferred.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Combination treatments may be used to providesignificant synergistic effects, particularly the combination of ananti-GM-CSFRα binding member with one or more other drugs. A bindingmember according to the present invention may be provided in combinationor addition to one or more of the following: NSAIDs (e.g. cox inhibitorssuch as Celecoxib and other similar cox2 inhibitors), corticosteroids(e.g. prednisone) and disease-modifying antirheumatic drugs (DMARDs)e.g. HUMIRA® (adalimumab), methotrexate, ARAVA®, ENBREL® (Etanercept),REMICADE® (Infliximab), KINERET® (Anakinra), RITUXAN® (Rituximab),ORENCIA® (abatacept), gold salts, antimalarials, sulfasalazine,d-penicillamine, cyclosporin A, diclofenac, cyclophosphamide andazathioprine.

In accordance with the present invention, compositions provided may beadministered to individuals. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and may depend on the severity of the symptomsand/or progression of a disease being treated. Appropriate doses ofantibody are well known in the art [28,29]. Specific dosages indicatedherein, or in the Physician's Desk Reference (2003) as appropriate forthe type of medicament being administered, may be used. Atherapeutically effective amount or suitable dose of a binding member ofthe invention can be determined by comparing its in vitro activity andin vivo activity in an animal model. Methods for extrapolation ofeffective dosages in mice and other test animals to humans are known.The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis or for treatment, the size and location ofthe area to be treated, the precise nature of the antibody (e.g. wholeantibody, fragment or diabody), and the nature of any detectable labelor other molecule attached to the antibody. A typical antibody dose willbe in the range 100 μg to 1 g for systemic applications, and 1 μg to 1mg for topical applications. Typically, the antibody will be a wholeantibody, preferably IgG1, IgG2 or more preferably IgG4. This is a dosefor a single treatment of an adult patient, which may be proportionallyadjusted for children and infants, and also adjusted for other antibodyformats in proportion to molecular weight. Treatments may be repeated atdaily, twice-weekly, weekly or monthly intervals, at the discretion ofthe physician. In preferred embodiments of the present invention,treatment is periodic, and the period between administrations is abouttwo weeks or more, preferably about three weeks or more, more preferablyabout four weeks or more, or about once a month. In other preferredembodiments of the invention, treatment may be given before, and/orafter surgery, and more preferably, may be administered or applieddirectly at the anatomical site of surgical treatment.

Binding members of the present invention will usually be administered inthe form of a pharmaceutical composition, which may comprise at leastone component in addition to the binding member. Thus pharmaceuticalcompositions according to the present invention, and for use inaccordance with the present invention, may comprise, in addition toactive ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. intravenous. Pharmaceutical compositionsfor oral administration may be in tablet, capsule, powder, liquid orsemi-solid form. A tablet may comprise a solid carrier such as gelatinor an adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. For intravenousinjection, or injection at the site of affliction, the active ingredientwill be in the form of a parenterally acceptable aqueous solution whichis pyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required. Binding members of the present invention may beformulated in liquid, semi-solid or solid forms depending on thephysicochemical properties of the molecule and the route of delivery.Formulations may include excipients, or combinations of excipients, forexample: sugars, amino acids and surfactants. Liquid formulations mayinclude a wide range of antibody concentrations and pH. Solidformulations may be produced by lyophilisation, spray drying, or dryingby supercritical fluid technology, for example. Formulations ofanti-GM-CSFRα will depend upon the intended route of delivery: forexample, formulations for pulmonary delivery may consist of particleswith physical properties that ensure penetration into the deep lung uponinhalation; topical formulations may include viscosity modifying agents,which prolong the time that the drug is resident at the site of action.In certain embodiments, the binding member may be prepared with acarrier that will protect the binding member against rapid release, suchas a controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare known to those skilled in the art. See, e.g., Robinson, 1978 [30].

Binding members according to the invention may be used in a method oftreatment or diagnosis of the human or animal body, such as a method oftreatment (which may include prophylactic treatment) of a disease ordisorder in a human patient which comprises administering to saidpatient an effective amount of a binding member of the invention.Conditions treatable in accordance with the present invention includeany in which GM-CSFRα plays a role. The published technical literatureindicates a role for GM-CSF in several diseases and conditions, assummarised below. Since GM-CSF binds specifically to GM-CSFRα,pathological and/or symptomatic effects of GM-CSF can be countered byinhibiting binding of GM-CSF to GM-CSFRα. Thus, the published evidence,in addition to the pharmacological in vivo and in vitro data presentedfor the antibody molecules described herein in the Experimental Part,indicates that binding members of the invention can be used in treatingautoimmune and/or inflammatory conditions, diseases and disorders, forexample rheumatoid arthritis, asthma, allergic response, multiplesclerosis, myeloid leukaemia and atherosclerosis. Published evidence onthese conditions is summarised below:

Asthma and Allergic Responses

Bronchial asthma is a common persistent inflammatory disorder of thelung characterised by airways hyper-responsiveness, mucusoverproduction, fibrosis and raised IgE levels. Airwayshyper-responsiveness (AHR) is the exaggerated constriction of theairways to non specific stimuli. Both AHR and mucus overproduction arethought to be responsible for the variable airway obstruction that leadsto the shortness of breath characteristics of asthma attacks(exacerbations) and which is responsible for the mortality associatedwith this disease (around 2000 deaths/year in the United Kingdom).

Recent studies have demonstrated that GM-CSF and its receptor areupregulated at both the protein and mRNA level in asthma. Furthermore,expression levels correlate to disease severity. Increased production ofGM-CSF has been measured in bronchoalveolar lavage (BAL) fluid, BALcells, sputum, bronchiolar epithelial cells, and antigen stimulatedperipheral blood mononuclear cells from asthma patients when compared tonon-asthmatic subjects [31, 32]. Furthermore, the level of airwayexpression of GM-CSF following allergen challenge has been shown tocorrelate with the degree of tissue eosinophilia and the severity of thelate phase asthmatic response [33]. Later studies linked upregulatedGM-CSFR expression to intrinsic or non-atopic asthma, correlating levelsof expression to lung function data [34]. In a mouse model of ovalbuminsensitisation and challenge, neutralisation of the activity of GM-CSFwith a goat polyclonal antibody, by intranasal administration prior toovalbumin challenge, prevented airways hyper-responsiveness and reducedboth the infiltration of eosinophils and mucus secretion into theairways [35]. Similarly in a mouse model of allergic respiratory diseaseinitiated by the intranasal administration of diesel exhaust particles,neutralisation of GM-CSF again by intranasal administration of a goatpolyclonal antibody prevented airways hyperresponsiveness tomethacholine, reduced BAL eosinophil counts and also diminished theexpression of mucus producing goblet cells on the airways epithelium[36].

The role of GM-CSF in allergic responses has been further investigatedin murine models of induced tolerance. Mice exposed to repeated dailydoses of nebulised ovalbumin without prior sensitisation developtolerance to ovalbumin and fail to elicit eosinophilic inflammation ofthe airways. Lung expression of GM-CSF via an adenoviral constructalters the responses of these animals and favours the influx ofeosinophils into the BAL, the generation of phenotypically allergichistology and associated goblet cell hyperplasia. This generation of atypical Th2 response is further evidenced by increased serum and BALconcentrations of IL-5 and serum IL-4. Further work in this model,utilising an MHC II KO mouse indicates that GM-CSF modulates theinteraction between antigen presenting cells and T cells in the airwaythereby facilitating T cell-mediated responses to ovalbumin [37].Significantly, the activity of GM-CSF as a potent activator of Th2responses can also be demonstrated in mice lacking IL-13 and/or IL-4,indicating that neutralisation of the activity of GM-CSF presents analternative therapeutic pathway distinct from the activity of thesecytokines.

Similar observations have been made in another murine model in whichrepeated intranasal exposure to ragweed results in Th2-typesensitisation and mild airway inflammation on re-exposure to antigen[38]. The administration of anti-GM-CSF antibodies in conjunction withragweed diminished Th2-associated cytokine production, presumably byinhibition of endogenous GM-CSF. In contrast, the delivery of ragweed toan airway microenvironment enriched with GM-CSF, either by multipleco-administrations of recombinant GM-CSF or a single delivery of anadenoviral vector carrying the GM-CSF transgene, resulted inconsiderably enhanced eosinophilic airway inflammation andragweed-specific Th2 memory responses.

Rheumatoid Arthritis (RA)

RA is a chronic inflammatory and destructive joint disease that affectsapproximately 1% of the population in the industrialised world. RA ischaracterised by hyperplasia and inflammation of the synovial membrane,inflammation within the synovial fluid, and progressive destruction ofthe surrounding bone and cartilage that commonly leads to significantdisability.

Whilst the cause of RA remains unknown, there is accumulating evidencefor the role of GM-CSF in the progression of RA. RA is believed to beinitiated and driven through a T-cell mediated, antigen-specificprocess. In brief, the presence of an unidentified antigen in asusceptible host is thought to initiate a T-cell response that leads tothe production of T-cell cytokines with consequent recruitment ofinflammatory cells, including neutrophils, macrophages and B-cells.

Many pro- and anti-inflammatory cytokines are produced in the rheumatoidjoint. Moreover, disease progression, reactivation and silencing aremediated via dynamic changes in cytokine production within the joint. Inparticular, TNF-α and IL-1 are considered to exert pivotal roles in thepathogenesis of RA and many of the newer therapies developed, or indevelopment, for the disease look to inhibit the activity of these twopro-inflammatory cytokines.

Recent studies in rodent models have suggested a central andnon-redundant role for GM-CSF in the development and progression of RA.Administration of exogenous recombinant GM-CSF enhances pathology in twodifferent mouse models of RA collagen-induced arthritis (CIA) [39] and amonoarticular arthritis model [40]. In addition to this is has beendemonstrated that GM-CSF knockout (GM-CSF−/−) mice are resistant to thedevelopment of CIA and that the levels of IL-1 and tumor necrosis factor(TNFα) found in synovial joint fluid was reduced compared to wildtypemice [41, 42]. Similarly, induction of monoarthritis usingintra-articular injection of methylated bovine serum albumin and IL-1 inGM-CSF^(−/−) mice results in reduced disease severity compared towild-type mice [43].

Furthermore, administration of murine anti-GM-CSF mAb significantlyameliorates disease severity in CIA and monoarticular arthritis models.In the CIA model, mAb treatment was effective in treating progression ofestablished disease, histopathology and significantly lowering jointIL-1 and TNF-α levels. In addition, mAb treatment prior to arthritisonset lessened CIA disease severity [44, 43].

A number of studies have analysed the levels of cytokines and receptorspresent in arthritic synovial fluid and membrane biopsy samples fromhuman tissue. Circulating mononuclear cells in 27 RA patients, 13healthy volunteers and 14 patients with osteoporosis were assessed forGM-CSFR levels by using PE-labelled GM-CSF [45]. In this study it wasdemonstrated that twice as many receptor positive cells were detected inRA patients (53%), compared to healthy controls (20%) and patientsundergoing investigation for osteoporosis (25%), thus suggesting thatmonocytes may be primed to respond to locally produced GM-CSF. Cytokinegene expression from RA patients [46] using in situ hybridization of SFcells demonstrated elevated levels of GM-CSF, IL-1, TNF-α and IL-6.Furthermore, isolated and cultured fibroblast-derived synoviocytes fromnormal volunteers demonstrated elevated protein levels of GM-CSF inresponse to IL-1α, IL-1β, TNF-α and TNF-β [47]. Quantification of serumlevels of GM-CSF in RA patients [48] showed that levels of protein wereincreased in severe (366 pg/ml, n=26) and moderate (376 pg/ml, n=58) RApatients compared to the control group (174 pg/ml, n=43), furthermore itwas also shown that GM-CSF was significantly elevated in the SF ofpatients with RA (1300 pg/ml).

Previously it has been observed that administration of recombinantGM-CSF in patients being treated for neutropenia could cause anexacerbation of RA [49]. Similar observations were made for a patientwith Felty's syndrome following treatment with recombinant GM-CSF [50].

Chronic Obstructive Pulmonary Disease (COPD)

Chronic Obstructive Pulmonary Disease (COPD) is defined as a diseasestate characterised by airflow limitation that is not fully reversible.The chronic airflow limitation is usually both progressive andassociated with an abnormal inflammatory response of the lungs tonoxious particles or gases. This airflow limitation is caused by amixture of small airway disease (obstructive bronchiolitis) andparenchymal destruction (emphysema), the relative contributions of whichvary from person to person. The resulting characteristic symptoms ofCOPD are cough, sputum production, and dyspnoea upon exertion. COPD is amajor public health problem and is the fourth leading cause of chronicmorbidity and mortality in the US. The disease is currently treated withdrugs originally developed for asthma such as oral or inhaledcorticosteroids with or without bronchodilators including P agonists.However, none of these drugs has been shown to slow the progression ofCOPD [51]. For example, corticosteroids which markedly suppress theeosinophilic inflammation in asthma do not appear to have any effect onthe inflammation seen in COPD which is predominantly neutrophil mediated[52]. Therefore, there is a need to develop new treatments for COPDwhich specifically target the inflammatory processes underlying thepathophysiology of this disease. GM-CSF, through its role in neutrophiland macrophage function, may play an important role in the pathogenesisof COPD.

In a study using guantitative PCR it was shown that in age matched COPDsputum versus non-obstructed smoker sputum GMCSF copy number wassignificantly elevated [53]. Furthermore, in a rodent model of cigarettesmoke induced lung inflammation, animals treated intranasally with anantibody to GM-CSF 2 days, 4hrs and 1hr prior to smoke exposuredemonstrated a significant reduction in neutrophils, macrophages andMMP-9 levels from the BAL when compared with the isotype controlantibody 5 days after challenge [54]. These studies are also supportedby our own observations investigating GM-CSF levels in induced sputumfrom patients with a range of COPD severities. In these studies weshowed that GM-CSF was elevated in the sputum of approximately 40% ofCOPD patients tested irrespective of disease severity, with GMCSF levelsapproaching 500pg/ml in some cases. GMCSF did not appear to be elevatedin non-smoking and smoking matched control patients. These data suggestthat GM-CSF may be one of the key mediators in smoke induced airwayinflammation and COPD.

Multiple Sclerosis (MS)

GM-CSF has been implicated in the autoimmune disease multiple sclerosis.By administering myelin oligodendrocyte glycoprotein (MOG) antigen torodents a model of human multiple sclerosis can be induced thatdemonstrates many of the phenotypes of MS such as central nervous systeminflammation and demyelination that can result in an MS like paralysis.In GM-CSF null mice MOG was unable to induce the EAE phenotype [55].Furthermore, it was shown that these mice had decreased T cellproliferation to MOG antigen and a decreased production of the Th1cytokines IL-6 and IFN-γ. Administration of GM-CSF neutralisingantibodies at the same time as antigen challenge prevented disease onsetfor 10 days after treatment with evidence of reduced lesions. Ifadministered after disease onset mice recovered completely within 20days of treatment [55].

Leukaemia

GM-CSF has also been implicated in the myeloid leukaemia, juvenilechronic myeloid leukaemia (JCML). This condition is a myeloproliferativedisorder that primarily affects patients less than 4 years of age. Invitro JCML peripheral blood granulocyte-macrophage progenitors (CFU-GM)demonstrate spontaneous proliferation at low cell densities, anobservation not previously described for other myeloproliferativedisorders. Furthermore, depletion of monocytes from these culturesabolished this proliferation. Subsequently it has been demonstrated thatthis spontaneous proliferation is mediated via a hypersensitivity of theJCML progenitors to the monocyte derived cytokine GM-CSF [56, 57, 58,59, 60, 61]. Rather than an overproduction or elevated levels of GM-CSFin JCML patients, the hypersensitivity of the JCML progenitors appearsto be through a deregulated GM-CSF induced Ras signal transductionpathway [62]. Recent studies with a GM-CSF analogue (E21R), thatantagonises the action of GM-CSF in both binding studies and functionalassays, has shown that by inhibiting the action of GM-CSF one cansignificantly reduce the JCML cell load in a severe combinedimmunodeficient/non obese diabetic (SCID/NOD) mouse xenograft model ofJCML [63]. Prophylactic systemic dosing of E21R at the time ofengraftment prevented JCML progenitors establishing in the bone marrowand dosing E21R 4 weeks post engraftment induced remission of JCML, witha reduction in cell load. Furthermore, administration of E21R toSCID/NOD mice co-engrafted with normal human bone marrow and JCML bonemarrow caused a reduction in JCML load however normal bone marrow cellsremained unaffected.

Atherosclerosis

Ischemic heart disease is the commonest cause of death worldwide. Overrecent years the concept that inflammation plays a significant role inthe pathogenesis of atherosclerosis has increased, with inflammatorycell accumulation occurring hand in hand with lipid accumulation in theartery walls.

Once resident in the arterial wall inflammatory cells, such as monocytesand macrophages, participate and perpetuate the local inflammatoryresponse. These macrophages also express scavenger receptors for a rangeof lipoproteins and thus contribute to the cells differentiation into‘foamy cells’. It is the death of these ‘foamy cells’ that contribute tothe development of the lipid core, a classic feature of these lesions.As the inflammation continues within these atherosclerotic plaques theseactivated inflammatory cells release fibrogenic mediators and growthfactors that promote smooth muscle cell (SMC) proliferation and fibrosisof the plaque. In addition to promoting fibrosis these cells alsorelease proteolytic enzymes, such as matrix metalloproteinase's (MMPs),that contribute to a weakening of the fibrotic plaque, thus renderingthem prone to disruption. These plaques once ruptured release cell debriand coagulation factors, such as tissue factor, into the vesselstimulating the coagulation cascade and development of thrombi. Theresulting arterial thrombosis can then lead to myocardial ischemia orinfarction.

Recently GM-CSF has been implicated in many aspects of diseaseprogression in atherosclerosis. In atherosclerotic lesions ofcholesterol fed rabbits GM-CSF was found to be co-localised withmacrophages and to a lesser degree endothelial cells and SMC [64].Furthermore, it has also been shown that GM-CSF expression is augmentedin human atherosclerotic vessels at the sites of macrophage accumulationand within medial SMCs and endothelial cells [65]. This increase inGM-CSF levels is, in part, attributed to the direct cell-cell contact ofmonocyte/macrophages and endothelial cells during the formation andpathogenesis of the atherosclerotic lesion [66]. Another key element inthe atherotic lesion is the ‘foamy cell’, that is macrophages that havetaken up oxidised low density lipoproteins (LDL) via scavenger receptorson the surface. In vitro this uptake of Ox-LDL can further stimulatemacrophages to proliferate via a GM-CSF dependent mechanism [67].

As atherosclerosis is a chronic inflammatory process anti-inflammatoryagents such as glucocorticoids have been investigated. Dexamethasone, ananti-inflammatory glucocorticoid, suppresses the development ofatherosclerosis in various experimental animal models [68,69,70,71]. Theefficacy of which has been attributed to inhibition of SMC migration[72] and proliferation [73], and reduction in the chemotaxis ofcirculating monocytes and leukocytes [74]. Recent studies shown thatox-LDL can induce GM-CSF release from mouse peritoneal macrophages [75].Furthermore, following treatment with dexamethasone this GM-CSF releasewas dose dependently inhibited, suggesting that the anti-inflammatoryaffects of dexamethasone are mediated by inhibition of the ox-LDLinduced GM-CSF production. As GM-CSF appears to have a central role inatherosclerosis, an alternative to glucocorticoids could be to inhibitthe GM-CSF activity in this indication.

Terminology

“And/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

GM-CSFRα and GM-CSF

GM-CSFRα is the alpha chain of the receptor for granulocyte macrophagecolony stimulating factor. The full length sequence of human GM-CSFRα isdeposited under Accession number S06945 (gi:106355) [76] and is set outherein as SEQ ID NO: 202. The mature form of human GM-CSFRα, i.e. withthe signal peptide cleaved, is set out herein as SEQ ID NO: 206. Unlessotherwise indicated by context, references herein to GM-CSFRα refer tohuman or non-human primate (e.g. cynomolgus) GM-CSFRα, normally human.GM-CSFRα may be naturally occurring GM-CSFRα or recombinant GM-CSFRα.

The 298 amino acid extracellular domain of human GM-CSF receptor α hasamino acid sequence SEQ ID NO: 205.

Unless otherwise indicated by context, references herein to GM-CSF referto human or non-human primate (e.g. cynomolgus) GM-CSF, normally human.

GM-CSF normally binds to the extracellular domain (SEQ ID NO: 205) ofthe mature GM-CSF receptor alpha chain (SEQ ID NO: 206). As describedelsewhere herein, this binding is inhibited by binding members of theinvention.

Naturally occurring splice variants of GM-CSFRα have been identified—seefor example refs. [77 and 78]. The extracellular domain is highlyconserved in these splice variants. Binding members of the invention mayor may not bind to one or more splice variants of GM-CSFRα, and may ormay not inhibit GM-CSF binding to one or more splice variants ofGM-CSFRα.

Binding Member

This describes a member of a pair of molecules that bind one another.The members of a binding pair may be naturally derived or wholly orpartially synthetically produced. One member of the pair of moleculeshas an area on its surface, or a cavity, which binds to and is thereforecomplementary to a particular spatial and polar organisation of theother member of the pair of molecules. Examples of types of bindingpairs are antigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. The present invention is concernedwith antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen-bindingsite. For example, a binding member may be an antibody molecule or anon-antibody protein that comprises an antigen-binding site. An antigenbinding site may be provided by means of arrangement of CDRs onnon-antibody protein scaffolds such as fibronectin or cytochrome B etc.[80,81,82], or by randomising or mutating amino acid residues of a loopwithin a protein scaffold to confer binding to a desired target.Scaffolds for engineering novel binding sites in proteins have beenreviewed in detail [82]. Protein scaffolds for antibody mimics aredisclosed in WO/0034784 in which the inventors describe proteins(antibody mimics) that include a fibronectin type III domain having atleast one randomised loop. A suitable scaffold into which to graft oneor more CDRs, e.g. a set of HCDRs, may be provided by any domain memberof the immunoglobulin gene superfamily. The scaffold may be a human ornon-human protein.

An advantage of a non-antibody protein scaffold is that it may providean antigen-binding site in a scaffold molecule that is smaller and/oreasier to manufacture than at least some antibody molecules. Small sizeof a binding member may confer useful physiological properties such asan ability to enter cells, penetrate deep into tissues or reach targetswithin other structures, or to bind within protein cavities of thetarget antigen.

Use of antigen binding sites in non-antibody protein scaffolds isreviewed in ref. [79]. Typical are proteins having a stable backbone andone or more variable loops, in which the amino acid sequence of the loopor loops is specifically or randomly mutated to create anantigen-binding site having for binding the target antigen. Suchproteins include the IgG-binding domains of protein A from S. aureus,transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type IIIdomain) and lipocalins. Other approaches include synthetic “Microbodies”(Selecore GmbH), which are based on cyclotides—small proteins havingintra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, abinding member according to the present invention may comprise otheramino acids, e.g. forming a peptide or polypeptide, such as a foldeddomain, or to impart to the molecule another functional characteristicin addition to ability to bind antigen. Binding members of the inventionmay carry a detectable label, or may be conjugated to a toxin or atargeting moiety or enzyme (e.g. via a peptidyl bond or linker). Forexample, a binding member may comprise a catalytic site (e.g. in anenzyme domain) as well as an antigen binding site, wherein the antigenbinding site binds to the antigen and thus targets the catalytic site tothe antigen. The catalytic site may inhibit biological function of theantigen, e.g. by cleavage.

Although, as noted, CDRs can be carried by scaffolds such as fibronectinor cytochrome B [80, 81, 82], the structure for carrying a CDR or a setof CDRs of the invention will generally be of an antibody heavy or lightchain sequence or substantial portion thereof in which the CDR or set ofCDRs is located at a location corresponding to the CDR or set of CDRs ofnaturally occurring VH and VL antibody variable domains encoded byrearranged immunoglobulin genes. The structures and locations ofimmunoglobulin variable domains may be determined by reference to(Kabat, et al., 1987 [98], and updates thereof, now available on theInternet (http://immuno.bme.nwu.edu or find “Kabat” using any searchengine).

Binding members of the present invention may comprise antibody constantregions or parts thereof, preferably human antibody constant regions orparts thereof. For example, a VL domain may be attached at itsC-terminal end to antibody light chain constant domains including humanCκ or Cλ chains, preferably Cλ chains. Similarly, a binding member basedon a VH domain may be attached at its C-terminal end to all or part(e.g. a CH1 domain) of an immunoglobulin heavy chain derived from anyantibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotypesub-classes, particularly IgG1, IgG2 and IgG4. IgG1, IgG2 or IgG4 ispreferred. IgG4 is preferred because it does not bind complement anddoes not create effector functions. Any synthetic or other constantregion variant that has these properties and stabilizes variable regionsis also preferred for use in embodiments of the present invention.

Binding members of the invention may be labelled with a detectable orfunctional label. Detectable labels include radiolabels such as ¹³¹I or⁹⁹Tc, which may be attached to antibodies of the invention usingconventional chemistry known in the art of antibody imaging. Labels alsoinclude enzyme labels such as horseradish peroxidase. Labels furtherinclude chemical moieties such as biotin that may be detected viabinding to a specific cognate detectable moiety, e.g. labelled avidin.Thus, a binding member or antibody molecule of the present invention canbe in the form of a conjugate comprising the binding member and a label,optionally joined via a linker such as a peptide. The binding member canbe conjugated for example to enzymes (e.g. peroxidase, alkalinephosphatase) or a fluorescent label including, but not limited to,biotin, fluorochrome, green fluorescent protein. Further, the label maycomprise a toxin moiety such as a toxin moiety selected from a group ofPseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof),Diptheria toxin (a cytotoxic fragment or mutant thereof), a botulinumtoxin A through F, ricin or a cytotoxic fragment thereof, abrin or acytotoxic fragment thereof, saporin or a cytotoxic fragment thereof,pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1or a cytotoxic fragment thereof. Where the binding member comprises anantibody molecule, the labelled binding member may be referred to as animmunoconjugate.

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antibody antigen-binding site. Antibody fragments thatcomprise an antibody antigen-binding site are molecules such as Fab,F(ab′)₂, Fab′, Fab′-SH, scFv, Fv, dAb, Fd; and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that retain the specificity of the original antibody.Such techniques may involve introducing DNA encoding the immunoglobulinvariable region, or the CDRs, of an antibody to the constant regions, orconstant regions plus framework regions, of a different immunoglobulin.See, for instance, EP-A-184187, GB 2188638A or EP-A-239400, and a largebody of subsequent literature. A hybridoma or other cell producing anantibody may be subject to genetic mutation or other changes, which mayor may not alter the target binding of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any binding member orsubstance having an antibody antigen-binding site. Thus, this termcovers antibody fragments and derivatives, including any polypeptidecomprising an antibody antigen-binding site, whether natural or whollyor partially synthetic. Chimeric molecules comprising an antibodyantigen-binding site, or equivalent, fused to another polypeptide aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023, and a large body ofsubsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. Human andhumanised antibodies are preferred embodiments of the invention, and maybe produced using any suitable method. For example, human hybridomas canbe made [83]. Phage display, another established technique forgenerating binding members has been described in detail in manypublications such as ref. [83] and WO92/01047 (discussed further below).Transgenic mice in which the mouse antibody genes are inactivated andfunctionally replaced with human antibody genes while leaving intactother components of the mouse immune system, can be used for isolatinghuman antibodies [84]. Humanised antibodies can be produced usingtechniques known in the art such as those disclosed in for exampleWO91/09967, U.S. Pat. No. 5,585,089, EP592106, U.S. Pat. No. 565,332 andWO93/17105. Further, WO2004/006955 describes methods for humanisingantibodies, based on selecting variable region framework sequences fromhuman antibody genes by comparing canonical CDR structure types for CDRsequences of the variable region of a non-human antibody to canonicalCDR structure types for corresponding CDRs from a library of humanantibody sequences, e.g. germline antibody gene segments. Human antibodyvariable regions having similar canonical CDR structure types to thenon-human CDRs form a subset of member human antibody sequences fromwhich to select human framework sequences. The subset members may befurther ranked by amino acid similarity between the human and thenon-human CDR sequences. In the method of WO2004/006955, top rankinghuman sequences are selected to provide the framework sequences forconstructing a chimeric antibody that functionally replaces human CDRsequences with the non-human CDR counterparts using the selected subsetmember human frameworks, thereby providing a humanized antibody of highaffinity and low immunogenicity without need for comparing frameworksequences between the non-human and human antibodies. Chimericantibodies made according to the method are also disclosed.

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors [85,86].

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment [87, 88, 89] which consists of a VH or a VL domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site [90,91]; (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; [92]). Fv, scFv ordiabody molecules may be stabilised by the incorporation of disulphidebridges linking the VH and VL domains [93]. Minibodies comprising a scFvjoined to a CH3 domain may also be made [94].

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain [89]. VH dAbs occur naturally in camelids (e.g. camel, llama) andmay be produced by immunising a camelid with a target antigen, isolatingantigen-specific B cells and directly cloning dAb genes from individualB cells. dabs are also producible in cell culture. Their small size,good solubility and temperature stability makes them particularlyphysiologically useful and suitable for selection and affinitymaturation. A binding member of the present invention may be a dAbcomprising a VH or VL domain substantially as set out herein, or a VH orVL domain comprising a set of CDRs substantially as set out herein. By“substantially as set out” it is meant that the relevant CDR or VH or VLdomain of the invention will be either identical or highly similar tothe specified regions of which the sequence is set out herein. By“highly similar” it is contemplated that from 1 to 5, preferably from 1to 4 such as 1 to 3 or 1 or 2, or 3 or 4, amino acid substitutions maybe made in the CDR and/or VH or VL domain.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways[95], e.g. prepared chemically or from hybrid hybridomas, or may be anyof the bispecific antibody fragments mentioned above. Examples ofbispecific antibodies include those of the BITE™ technology in which thebinding domains of two antibodies with different specificity can be usedand directly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, directed againstGM-CSFRα, then a library can be made where the other arm is varied andan antibody of appropriate target binding selected. Bispecific wholeantibodies may be made by knobs-into-holes engineering [96].

Antigen-Binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that binds to and is complementary to all or part of thetarget antigen. Where an antigen is large, an antibody may only bind toa particular part of the antigen, which part is termed an epitope. Anantibody antigen-binding site may be provided by one or more antibodyvariable domains. Preferably, an antibody antigen-binding site comprisesan antibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

Kabat Numbering

Residues of antibody sequences herein are generally referred to usingKabat numbering as defined in Kabat et al., 1971 [97]. See also refs.[98, 99].

Isolated

This refers to the state in which binding members of the invention, ornucleic acid encoding such binding members, will generally be inaccordance with the present invention. Isolated members and isolatednucleic acid will be free or substantially free of material with whichthey are naturally associated such as other polypeptides or nucleicacids with which they are found in their natural environment, or theenvironment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo. Members and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themembers will normally be mixed with gelatin or other carriers if used tocoat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC85110503)) cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. pA₂ analysis of two anti-GM-CSFRα antibodies in the TF-1proliferation assay. Proliferation of TF-1 cells was induced withincreasing concentrations of GM-CSF in the presence of increasingconcentrations of two optimised IgG4, Antibody 6 (FIG. 1A) and Antibody1 (FIG. 1B), respectively. For data shown in graph 1A and graph 1B theincorporation of tritiated thymidine was measured and the EC50 of GM-CSFat each concentration of antibody was calculated. For data shown ingraph IC and graph 1D dose ratios were then calculated and analysed bySchild regression in order to obtain pA₂ values.

FIG. 2. pA₂ analysis of an anti-GM-CSFRα antibody, Antibody 6, in thegranulocyte shape change assays. Human (graph 2A and 2C) or cynomolgus(2B and 2D) granulocytes were treated with increasing concentrations ofGM-CSF in the presence of increasing concentrations of IgG4. The changein shape of the granulocytes was measured using flow cytometry and theEC50 of GM-CSF at each concentration of antibody was calculated (graph2A and graph 2B). Dose ratios were then calculated and analysed bySchild regression in order to obtain pA₂ values (graph 2C and graph 2D).

FIG. 3. Antagonist potency of two antibodies, Antibodies 1 and 6,respectively, as IgG4s in an assay measuring proliferation of TF-1 cellsinduced by 7 pM human GM-CSF. Also shown are data for positive controlIgG4 2B7 and for an isotype control IgG4. Data represent the mean withstandard deviation bars of triplicate determinations within the sameexperiment.

FIG. 4. Antagonist potency of two antibodies, Antibodies 1 and 6,respectively, as IgG4s in an assay measuring the shape change of humangranulocytes induced by 7 pM human GM-CSF. Also shown are data forcontrol IgG4 2B7 and for an isotype control IgG4. Data represent themean with standard deviation bars of triplicate determinations withinthe same experiment.

FIG. 5. Antagonist potency of two antibodies, Antibodies 1 and 6,respectively, as IgG4s in an assay measuring TNFα release from humanmonocytes stimulated with 1 nM human GM-CSF. Also shown are data forcontrol antibody 2B7 and for an isotype control IgG4. Data represent themean with standard deviation bars of triplicate determinations withinthe same experiment.

FIG. 6. Antagonist potency of two antibodies, Antibodies 1 and 6,respectively, as IgG4s in an assay measuring human granulocyte survivalinduced by 7 pM human GM-CSF. Also shown are data for the controlantibody 2B7 and for an isotype control IgG4. Data represent the meanwith standard deviation bars of triplicate determinations within thesame experiment.

FIG. 7. Affinity matured human mabs Antibody 1 and Antibody 6, but notthe parent human mAb 28G5 (Antibody 3) or the known murine antibody 2B7,inhibit GM-CSF driven differentiation of human hemopoietic progenitors.5×10⁴ thawed mononuclear cells from an apheresis sample were cultured insemi-solid agar in the presence of 10 ng/ml GM-CSF and the indicatedconcentration of mAb. Colonies were counted at day 14. Graph showsnumber of colonies against mAb concentration in μg/ml.

FIG. 8. Dose-response analysis of the efficacy of affinity matured mAbin huGM-CSFR Tg chimeric mice. Groups of 5 Tg chimeric mice were treatedwith 500 ng huGM-CSF (or PBS) s.c twice daily for 4 days (D.1-D.4) andeither control (CAT001) or test mAb (Antibody 6) at the indicatedconcentrations on D.0, Spleen weights were assessed on D.5.

FIG. 9. Dose-response analysis of the efficacy of Antibody 6 in a humanperipheral blood mononuclear cell endogenous cytokine release assay.1×10 ⁶ cells were cultured for 72 hrs in the presence and absence ofantibody and an IL-6 and TNFa ELISA performed on the supernatants. Datarepresent the mean inhibition with standard deviation bars of duplicatedeterminations within the same experiment.

EXPERIMENTAL PART Background

Human antibody fragments may be selected in vitro from repertoiresdisplayed on the surface of filamentous bacteriophage. This process isknown as phage display and provides a means of deriving human antibodyfragments. The process can be used to isolate human anti-humanspecificities and may be tailored to derive antibodies of particularaffinity characteristics.

Antibody fragments consisting only of the heavy chain variable (VH) andlight chain variable (VL) domains joined together by a short peptidelinker contain all the information that is necessary to determineantigen binding. Such fragments are known as single chain Fv (scFv).When displayed on the phage surface, scFv have been shown to both foldcorrectly and bind to antigen. Large repertoires of human scFv have beenconstructed in this way, and have provided a source from whichindividual clones may be isolated for development as drug candidates.Candidate scFv are then reformatted as whole IgG (typically human IgG)molecules for therapeutic applications.

Summary

Selections were carried out on an scFv phage display library derivedfrom human spleen lymphocytes in order to enrich for populations ofphage that bound to human GM-CSFRα. We isolated scFv antibodies havingselected characteristics and converted these scFv into IgG₄. Using avariety of assays, a panel of antibodies were isolated, optimised andgermlined to produce IgG4 with appropriate specification for atherapeutic antibody.

19 antibody clones, whose sequences are shown as antibodies 1, 2 and4-20 in the sequence listing, were derived from a parent antibody. Theparent is shown as antibody 3 in the sequence listing, and is alsoreferred to herein as 28G5. The 19 clones were selected as showingparticularly good properties in a range of biological assays, asdescribed in the Experimental Part, and were designated antibody numbers1, 2 and 4 to 20.

The bioassays were designed to reflect the inflammatory nature ofdiseases such as rheumatoid arthritis. For example, the shape change ofneutrophils necessary for their recruitment to the site of action, therelease of proinflammatory factors by monocytes and the increasedsurvival of inflammatory cell types in response to particular signals.The antibodies exhibit potent neutralisation activity in these assays.

Detailed protocols of the assay methods used are provided below in thesection entitled “Assay Materials and Methods”.

Antibody Lead Isolation

A large single chain FV (scFv) human antibody library was used forselections. This was derived from the spleen lymphocytes from 20 healthydonors and cloned into a phagemid vector. ScFv which recognised GM-CSFRαwere isolated from the phage display library in a series of repeatedselection cycles on purified GMCSF-RA derived from overexpression of apurification-tagged, soluble, extracellular domain of the receptor inHEK293T cells. This was achieved essentially as described in Vaughan etal [102]. In brief, following exposure of the biotinylated receptor tothe phage library, the protein with phage bound was captured onstreptavidin coated magnetic beads. Unbound phage were washed away.Bound phage were then rescued as described by Vaughan et al and theselection process was repeated. Three rounds of selection were carriedout at reducing antigen concentrations. A representative proportion ofscFvs from the output of selection rounds were subjected to DNAsequencing.

Following these initial selections from the phage display library, apanel of unique scFv were identified in a ligand binding assay, whichwas designed to identify phage expressing scFv antibodies that werecapable of inhibiting binding of GM-CSF to purified GM-CSFRαextracellular domain. Neutralising potency of these scFv in the ligandbinding assay ranged from 0.65 to 3.3 nM.

Antibodies that were active in the biochemical ligand binding assay wereassessed for biological activity in a TF-1 proliferation assay, whichmeasured neutralisation potency by assaying ability of the antibodies toinhibit the proliferation of TF-1 cells stimulated with GM-CSF. TF-1 isa human premyeloid cell line established from a patient witherythroleukemia. This cell line is factor-dependent for survival andproliferation and is routinely maintained in human GM-CSF. Inhibition ofGM-CSF dependent proliferation was determined by measuring the reductionin incorporation of tritiated thymidine into the newly synthesised DNAof dividing cells. All of the scFv had measurable potency in this assay,with IC50 values ranging from about 180 to 1200 nM.

The most potent scFv clones were reformatted as human IgG4 antibodymolecules with a human gamma 4 heavy chain constant domain and a humanlambda light chain constant domain. Vectors were constructed for themost potent scFv clones in order to allow expression of the antibodiesas whole IgG4 antibody as described by Persic et al. [100] with a fewmodifications. An oriP fragment was included in the vectors tofacilitate use with the HEK-EBNA 293 cells and to allow episomalreplication. The VH variable domain was cloned into the polylinkerbetween the secretion leader sequence and the human gamma 4 constantdomain of the expression vector pEU8.1(+). The VL variable domain wascloned into the polylinker between the secretion leader sequence and thehuman lambda constant domain of the expression vector pEU4.1(−).HEK-EBNA 293 cells were co-transfected with the constructs expressingheavy and light chain and whole antibody was purified from theconditioned media using protein A affinity chromatography. The purifiedantibody preparations were sterile filtered and stored at 4° C. inphosphate buffered saline (PBS) prior to evaluation. Proteinconcentration was determined measuring absorbance at 280 nm using theBCA method (Pierce).

The re-formatted IgG were compared to the known murine antibody 2B7 inthe TF-1 proliferation assay. The IgG4s retained or gained activity inthis assay, with IC50 values ranging from 6 to about 1600 nM.

In inflammatory disease, the shape change of neutrophils is necessaryfor their recruitment to the site of action. A human granulocyte shapechange assay was designed to mimic this biological response usingfluorescence activated cell sorting (FACS) to measure the change inshape of granulocytes isolated from blood following their exposure toGM-CSF. The ability of anti-GM-CSFRα IgG4 antibodies to inhibit theshape change response of neutrophils to GM-CSF was assessed, and IC50values of selected clones ranged from about 15 to 350 nM. Arepresentative antibody 28G5 neutralized cynomolgus GMCSF-R in thecynomolgus granulocyte shape change assay with an IC50 of about 5 nM.The known murine antibody 2B7 was also able to neutralise the biologicalresponse resulting from GM-CSF binding to the cynomolgus receptor.

Receptor binding affinity of the antibodies was then measured usingBIACORE®, with calculated K_(D) values ranging from 32 to 377 nM.

Optimisation

In an effort to improve the potency of 28G5 an optimisation programmewas initiated. Libraries of antibodies were produced where randommutagenesis of the V_(H) or V_(L) CDR3s was carried out. Each CDR3 wasrandomised in two blocks of 6 amino acids in order to cover the entireCDR, producing libraries H1 (N terminal block of 6 aa VH CDR3), H2 (Cterminal block of 6 aa in VH CDR3), L1 (N terminal block of 6 aa in VLCDR3) and L2 (C terminal block of 6 aa in VL CDR3). The resultinglibraries were subjected to repeated selection cycles for binding tohuman GM-CSFRα. Clones isolated from this selection process were thenused to construct a combined phage library which contained scFv withboth mutated heavy chain CDR3s and mutated light chain CDR3s. Theselibraries were also subjected to same selection procedure.

At each stage of the optimisation process, scFv that were able toinhibit the binding of 28G5 IgG4 to the GM-CSF receptor were identifiedusing an epitope competition assay with 28G5 and the receptor, and werethen assessed in the TF-1 proliferation assay, as described below.

Following random mutagenesis of heavy chain CDR3 sequences of 28G5, apanel of scFv were identified with improved potency in the TF-1proligeration assay. Most of the potency improvements were obtained whenthe 3′ end of the VH CDR3 was randomised.

Following random mutagenesis of light chain CDR3 sequences of 28G5, apanel of scFv were identified with measurable neutralisation potency inthe TF-1 assay. All of the potency improvements were obtained when the3′ end of the VL CDR3 was randomised.

Following combination of the heavy and light chain CDR3 randommutagenesis libraries, a panel of scFvs were identified with improvedpotency in the TF-1 proliferation assay over the parental scFv 28G5.ScFv with potency improvements of >60000 fold over parent 28G5 wereisolated. All combinations of the libraries resulted in improved scFv,ie H1/L1, H1/L2, H2/L1, H2/L2. This is of particular interest because noimproved scFvs were isolated from the L1 library.

A panel of 19 scFv identified during the optimisation of 28G5 werereformatted and expressed as IgG4s, using the methods described above.The panel was composed of antibody clones 1, 2 and 4 to 20. Some of themost potent clones in this panel were obtained from the combined H and LCDR3 mutagenised libraries. The IgG4 antibodies in this panel wereassessed for their activity in the TF-1 proliferation assay and werecompared to the known murine antibody 2B7. All of the optimised IgG4swere more potent than 2B7 in this assay. On this occasion 2B7 had acalculated IC50 of about 1.6 nM, whereas the clones had calculated IC50values ranging from about 1 pm to about 1100 pM. Data are presented inTable 1 below and summarised as follows:

-   IC50<1500 pM Antibodies 1, 2 and 4 to 20-   IC50<300 pM Antibodies 1, 2, 4-12 and 14-20-   IC50<60 pM Antibodies 1, 2, 4-6, 8-11, 14 and 16-20-   IC50<10 pM Antibodies 1, 5, 6, 11 and 20.

FIG. 3 illustrates antagonist potency of two representative antibodiesof the invention, Antibody 1 and Antibody 6, in comparison with theknown antibody 2B7 in the TF-1 proliferation assay.

The BIACORE®2000 System (Pharmacia Biosensor) was used to assess thekinetic parameters of the interaction of some of the lead-optimisedIgG4s with recombinant purification-tagged GM-CSF receptor extracellulardomain. The affinity of the antibodies was much improved, withcalculated K_(D) values from 0.127 nM to about 5 nM. Data are shown inTable 2. Improvements were obtained in both on-rates and off rates. Thecorrelation between the affinity of the IgG4s for the solubleextracellular domain of GM-CSFR α and their performance in the TF-1assay was very good with a Pearson coefficient of 0.85 (p<0.0001). Byway of comparison, KD of 2B7 was separately calculated and was shown tobe about 7 nM.

IgG4 antibodies identified during the optimisation of 28G5 were assessedin the human granulocyte shape change assay and were compared to theknown murine antibody 2B7. All of the antibodies that were assessed inthis assay (antibodies 1, 2, 5, 6, 9-11, 16 and 20) were very potentwith IC50s ranging from 7.8 to 90 pM. Of these, antibodies 1, 2, 5, 6,9, 16 and 20 had IC50s less than 50 pM, and antibodies 1, 2, 6, 16 and20 had IC50s less than 25 pM. Our antibodies were more potent than 2B7,which had an IC50 of 477 pM. Data are shown in Table 3. FIG. 4illustrates antagonist potency of two representative antibodies of theinvention, Antibody 1 and Antibody 6, in comparison with the knownantibody 2B7 in the human granulocyte shape change assay.

IgG4 antibodies identified during the optimisation of 28G5 were assessedin the cynomolgus granulocyte shape change assay. All of the antibodieswere able to neutralise the activity of GM-CSF at the cynomolgusreceptor as well as at the human receptor and all of the antibodies weremore potent than 2B7. 2B7 had an IC50 of 26 pM whereas representativeantibodies (Antibody 6, Antibody 1 and Antibody 2) from the panel hadIC50 values of 1.73, 2.03 and 3.2 pM, respectively.

A panel of the IgG4s identified during the optimisation of 28G5 wereassessed for their neutralisation potency in the monocyte TNFα releaseassay. This assay tests for ability to inhibit release of theproinflammatory factor TNFα from human monocytes when they are treatedwith GM-CSF. Antibodies 1, 2, 5, 6, 9 and 10 were tested and all wereactive in this assay and were able to fully neutralise the action ofGM-CSF at its receptor (IC50 ranging from about 43 to 139) whereas at aconcentration of 333 nM 2B7 could only achieve 50% inhibition of GM-CSFinduced TNFα release, indicating that this antibody is only a partialinhibitor in this assay. FIG. 5 illustrates antagonist potency of tworepresentative antibodies of the invention in comparison with the knownantibody 2B7 in the monocyte TNFα release assay. Data are shown in Table4 and are summarised as follows:

-   <150 pM Antibody nos 1, 2, 5, 6, 9 & 10-   <110 pM Antibody nos 1, 2, 5, 6 & 9 <100 pM Antibody nos 1, 5, 6 & 9

A hallmark of inflammatory disease is the enhanced survival ofinflammatory cell types in response to particular signals. Granulocytesare able to survive for longer in the presence of GM-CSF and so theability of the IgG4 antibodies isolated during the optimisation of 28G5to inhibit this response was assessed in a granulocyte survival assay.All of the anti-GM-CSFRα IgG4s from lead optimisation were active inthis assay, and representative neutralisation potencies (IC50) rangedfrom 7.0 to 843.7 pM. This is in contrast to the known murine antibody2B7 which was completely inactive up to a concentration of 83 nM. FIG. 6illustrates antagonist potency of two representative antibodies of theinvention, Antibody 1 and Antibody 6, in comparison with the knownantibody 2B7 in the granulocyte survival assay.

These data, as illustrated in FIGS. 3 to 6, indicate that our antibodieshave significantly different properties compared with the known murineantibody 2B7. For example, representative antibodies of the inventioninhibited granulocyte survival and TF-1 proliferation stimulated with 7pM GM-CSF in the granulocyte survival and TF-1 proliferation assaysrespectively, whereas 2B7 did not inhibit granulocyte survival but didinhibit TF-1 proliferation (albeit to a lesser extent than ourantibodies). The data indicate that binding members of the inventionhave higher affinity and improved ability to inhibit a variety ofbiological effects mediated through GM-CSF-R compared with knownanti-GM-CSFRα antibodies.

The derived amino acid sequence of 28G5 and its derivatives were alignedto the known human germline sequences in the VBASE database and theclosest germline identified by sequence similarity. The closest germlinefor the VH domain of 28G5 and its derivatives was identified as VH1 DP5.The 28G5 VH has 14 changes from the VH1-24 (DP5) germline withinframework regions.

The closest germline for the VL domain is Vlambdal VL 1-e (DPL8), whichhas only 5 changes from the germline within the framework regions.Framework regions of 28G5 and its derivatives were returned to germlineby site directed mutagenesis to identically match native humanantibodies. All except one amino acid could be converted to germlinewith only modest changes in antibody potency. The amino acid isoleucineat position 94 of the heavy chain (using Kabat numbering, Kabat et al.1971) could not be changed to the germline threonine without a completeloss of activity. This single change from germline was thereforemaintained in the antibody framework region.

A full pA₂ analysis of two of the anti-GM-CSFRα antibodies, Antibody 6and Antibody 1, was carried out in the TF-1 proliferation assay. Thedata confirms that these antibodies are highly potent antagonists inthis system with calculated pA₂ values of −11.3±0.2 and −11.0±0.2respectively (FIG. 1).

A full pA₂ analysis of one of the anti-GM-CSFRα antibodies, Antibody 6,was carried out in the human and cynomolgus granulocyte shape changeassays. The data confirm that this antibody is a highly potentantagonist in these systems with calculated pA₂ values of −10.58 and−10.78 in the human and cynomolgus assays respectively (FIG. 2).

GM-CSF drives the differentiation of haemopoietic progenitor cells intogranulocyte and macrophage colonies in semi-solid agar assays. Affinitymatured Antibody 6 and Antibody 1, the parent mAb Antibody 3 (28G5) anda negative control (CAT001) were therefore assessed for their ability toantagonise this GM-CSF specific activity using progenitor cells derivedfrom peripheral blood, in a colony formation assay. Data presented inFIG. 7 demonstrates that both affinity matured representative mabs werepotent inhibitors of in vitro haemopoietic colony formation mediated byhuman GM-CSF.

Approximate IC₅₀ values were 0.08 μg/ml (Antibody 6) and 0.25 μg/ml(Antibody 1) for the affinity matured mAb. Interestingly the knownmurine antibody 2B7 appeared to have little if any inhibitory activityin this assay up to a concentration of 66 nM.

In control experiments the affinity matured mAb had no effect on colonyformation mediated by the combination of SCF+IL-3+G-CSF as expected and,in the absence of cytokines, colony formation was negligible (<4colonies/culture).

For in vivo analysis of huGM-CSFRα specific mAb antagonist activity,transplantation of bone marrow from transgenic (Tg) mice expressing boththe α and the β chains of human GM-CSFR into wildtype mice can be usedto generate chimeric animals such that transgenic huGM-CSFR expressionis limited to bone marrow derived haemopoietic cells and thus moreclosely resembles the expression profile of the endogenous receptor. Inthese Tg chimeric mice the administration of huGM-CSF leads to anincrease in spleen weight and the marginalisation of circulating bloodmonocytes. Affinity matured Antibody 6 and a negative control mAb,CAT001 were assessed for their ability to antagonise these GM-CSFmediated in vivo responses. For dose-response analysis 6 groups of 5 Tgchimeric mice were treated with 500 ng huGM-CSF s.c twice daily for 4days (day 1-4) and a seventh control group of five animals received PBSonly. Four of the 6 groups of huGM-CSF treated animals received test mAb(Antibody 6) at 16 mg/kg, 5.3 mg/kg, 1.78 mg/kg or 0.59 mg/kg at D.0while a fifth group of the huGM-CSF treated animals received controlCAT001 at 16 mg/kg at D.0. Results presented in FIG. 8 demonstrate that,compared with control PBS, treatment with huGM-CSF induced a significantincrease in spleen weight and a decrease in circulating blood monocytes.As expected, treatment with 16 mg/kg control CAT001 had no effect oneither the increase in spleen weight or the decrease in blood monocytes.In contrast there was a clear dose-response effect following treatmentwith the test mAb Antibody 6, as at 16 mg/kg this antibody abolished theincrease in spleen weight and, while still apparent, the effect wasgreatly reduced at 0.59 mg/kg of mAb. The IC₅₀ would appear to besomewhere between 0.59 mg/kg and 1.78 mg/kg. A similar result wasobserved for the GM-CSF induced decrease in circulatingmonocytes—treatment with test mAb Antibody 6 at 16 mg/kg abolished thedecrease, while mAb at 0.59 mg/kg had only a minor impact on thisresponse. These data show that the anti-GM-CSFRα antibody is anantagonist of human GM-CSFRα in vivo.

To further investigate the anti-inflammatory properties of theseanti-GM-CSFRα antibodies, Antibody 6 was evaluated in a peripheral bloodmononuclear cell cytokine release assay. In this assay TNFα and IL-6 canbe endogenously released depending on the donor. In this assay theGM-CSF is also endogenously produced by the cells, rather thanexogenously added, and therefore results observed in this assayrepresent inhibition of the biological effects of native endogenousGM-CSF binding to its receptor.

Following administration of antibody 6 both these cytokines were dosedependently inhibited as illustrated in FIG. 9. These data indicate thatthese antibodies can inhibit the activity of native GM-CSF and that byinhibiting GM-CSF signalling one can inhibit key pro-inflammatorycytokines, such as IL-6 and TNFα, both of which being implicated in anumber of inflammatory indications such as rheumatoid arthritis.

Furthermore, based on this result with Antibody 6 it can be expectedthat each of antibodies 1 to 20 would also demonstrate inhibition inthis assay, since all of antibodies 1 to 20 are believed to bind thesame region of GM-CSFRα.

Mapping of Residues Important for Antigen Recognition, and SequenceAnalysis

We determined the variability of residues at positions in the germlinedAntibody 6 scFv sequence in order to identify which positions arenormally conserved for ligand binding and which positions are variablein an antibody that still retains ligand binding activity.

Positions contributing to antigen binding appeared to be Kabat residues27A, 27B, 27C, 32, 51, 52, 53, 90, 92 and 96 in the VL domain and Kabatresidues 17, 34, 54, 57, 95, 97, 99 and 100B in the VH domain.

Seven positions that appeared to be important for antigen binding wereidentified: H95, H97, H99, H100B, L90, L92 and L96. We then analysed theresidues at these positions in sequences of 160 variants isolated duringthe 28G5 antibody optimisation process, all of which showed a minimum5-fold improvement in potency in the TF-1 proliferation assay.

Data in Table 5 below summarise the different amino acids (out of apossible 20) that were observed in each of these positions, and at L95A.Where positions are strongly conserved to the amino acids present in28G5 and/or Antibody 6, this is good evidence that those amino acids arekey to binding the antigen. For example, the residues at the followingpositions are strongly conserved: H97, H100B, L90, L92.

Method

The DNA sequence encoding the affinity matured and germlined Antibody 6scFv was converted to ribosome display format, essentially as describedin ref. [101]. Error prone PCR was performed on the Antibody 6 sequence,using the high mutation conditions (7.2 mutations per 1,000 bp) in themanufacturer's protocol (BD Bioscience), in order to create a library ofvariant 574D04 sequences containing random point mutations. This librarywas expressed on ribosomes and incubated with purification-taggedGM-CSFRα to allow binding to occur. Variants able to bind to taggedGM-CSFRα were captured and removed using paramagnetic beads coated withprotein G (Dynal). The unbound variants remaining in the population wereadded to a pool of four biotinylated anti-idiotypic antibodies, whichhad previously been derived from the large human antibody phage displaylibrary described in ref. [102] and were known to bind to the Antibody 6scFv. Variants bound by the biotinylated anti-idiotypic antibodies werecaptured with streptavidin beads whilst unbound variants were washedaway. This process was repeated for two further rounds of ribosomedisplay selection, following the general methodology of ref. [101].

A representative proportion of variants from the selection outputs wascloned into a phagemid vector and the scFv variants were expressed onphage for testing by ELISA, using the same method as described inEdwards B M et al (2003) Journal of Molecular Biology Vol 334:103. Thosevariants that did not display binding to purification-tagged GM-CSFRαwere tested for binding to the pool of four anti-idiotypic antibodieswhich were used in the selection. Variants which, in the anti-idiotypebinding assay, demonstrated binding which was equal to or greater thanthe Antibody 6 scFv were sequenced and the sequences were analysed tofind positions at which there was a high frequency of mutation.

The average mutation rate of the population of variants was found to be3.05 amino acids per V_(H) or V_(L) chain, using 486 sequences for theV_(H) chains and 451 sequences for the V_(L) chains. They were analysedfor mutational hotspots, plotting frequency of mutation in relation totheir position along the scFv. The analysis focussed on those cloneswith at least one CDR mutation per V_(H) and V_(L) and less than 4mutations per V_(H) and V_(L). From this panel of 123 V_(H) and 148V_(L) sequences, hotspots were defined as those that had a mutationalfrequency of 5% or more.

Seven positions within V_(H)CDR3 and V_(L)CDR3 of Antibody 6 werehighlighted as putative positions important for antigen binding usingthe ribosome display negative selection method. An analysis was thenperformed on 160 sequence variants isolated during the 28G5 antibodyoptimisation process, in which the entire V_(H)CDR3 and V_(L)CDR3sequences were randomised and selected for higher affinity. Allsequences (including Antibody 6) are variants of 28G5 which showed aminimum 5-fold improvement in potency in the TF-1 proliferation assay.

Determination of Linear Epitope

We screened Antibody 6 and the known antibody 2B7 against 2442 peptides,each representing short regions of amino acid sequence from theextracellular portion of GM-CSFR-α, using a PEPSCAN method. Bindingsignals for each antibody against all the peptides were averaged togenerate a mean background signal and for each peptide asignal/background ratio was calculated. For both Antibody 6 and 2B7 asignal/background ratio of four or greater was counted as a specific,positive signal. The sequences of peptides giving a specific, positivesignal were analysed for conserved binding motifs and it was found thatAntibody 6 bound preferentially to a YLDFQ motif, corresponding toresidues 226 to 230 of mature human GM-CSFRα, and the 2B7 antibody boundpreferentially to a DVRI motif, corresponding to residues 278 to 281 ofmature human GM-CSFRα. Amino acid sequence numbering for the maturereceptor is as set out in SEQ ID NO: 206.

PEPSCAN Method (Peptide-Binding Scan)

Overlapping mostly 15-mer synthetic peptides having sequences derivedfrom GMCSF were synthesized and screened using credit-card formatmini-PEPSCAN cards (455-well-plate with 3 μl wells) as describedpreviously [103]. Binding of antibodies to each peptide was tested in aPEPSCAN-based enzyme-linked immuno assay (ELISA). 455-well creditcard-format polypropylene cards containing the covalently linkedpeptides were incubated with sample (for example 10 μg/ml antibody orserum diluted 1/1000 in a PBS solution which contains 5% horse-serum(v/v) and 5% ovalbumin (w/v)) and 1% Tween80 or in case of mild blockingin a PBS solution with 4% horse-serum (v/v) and 1% Tween80 (4° C,overnight). After washing, the peptides were incubated with ananti-antibody peroxidase (dilution 1/1000, for example rabbit-anti-mouseperoxidase, Dako) (1 hr. 25° C), and subsequently, after washing theperoxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate(ABTS) and 2 μl/ml 3% H₂O₂ were added. After 1 hr the colour developmentwas measured. The colour development of the ELISA was quantified with aCCD-camera and an image processing system. The setup consists of aCCD-camera and a 55 mm lens (Sony CCD Video Camera XC-77RR, Nikonmicro-nikkor 55 mm f/2.8 lens), a camera adaptor (Sony Camera adaptorDC-77RR) and the Image Processing Software package Optimas, version 6.5(Media Cybernetics, Silver Spring, Md. 20910, U.S.A.). Optimas runs on apentium computer system.

Assay Materials and Methods

Biochemical Ligand Binding Assay

Purified scFv preparations were prepared as described in Example 3 ofWO01/66754 [104]. Protein concentrations of purified scFv preparationswere determined using the BCA method [105]. Fluoronunc™ 96 wellmicrotitre plates were coated overnight at 40° C. with 50 μl/well ofanti-human IgG4 diluted to 2.5 μg/ml in PBS. Plates were washed 3 timeswith 300 μl/well of PBS/0.1% Tween-20 before blocking for 1 hour at roomtemperature with 300 μl/well of 3% BSA in PBS. Plates were washed again3 times with 300 μl/well of PBS/0.1% Tween-20 and then 50 μl of humanGM-CSFRα diluted to 62.5 ng/ml in 1% BSA/PBS was added to each well andthe plates were incubated for 1 hour at room temperature. After washing3 times as described above, 25 μl of sample material was added to eachwell followed by 25 μl of biotinylated GM-CSF diluted to 2 nM in 1%BSA/PBS. To define total binding, buffer only was used as the samplematerial. To define non-specific binding, unlabelled GM-CSF diluted to100 nM in 1% BSA was used as the sample material. Plates were incubatedfor 1 hour at room temperature before washing 3 times as describedabove. 50 μl of europium labelled streptavidin (PerkinElmer) diluted to100 ng/ml in DELFIA™ assay buffer was added to each well of the plateand was incubated for 30-60 minutes at room temperature before washing 7times with DELFIA™ wash buffer. 50 μl/well of DELFIA™ enhancementsolution was added to the plates and the samples were read at 615 nm ona platereader.

TF-1 Proliferation Assay

TF-1 cells, obtained from R&D Systems and routinely maintained in RPMI1640, 10% FBS, 1 mM sodium pyruvate and 4 ng/ml GM-CSF, were starved bywashing 3 times in assay medium (RPMI 1640, 5% FBS, 1 mM sodiumpyruvate), resuspending in assay medium and incubating for 7-24 hours at37° C. in 5% CO₂. Cells were then resuspended at 1×10⁵/ml in assaymedium and 100 μl was added to each well of a 96 well flat-bottomedtissue culture plate. Test samples were prepared by sterile filteringthe stock sample prior to diluting in assay medium. ═μl of test materialwas then added to each well of cells and these were incubated for 45-60mins at 37° C. in 5% CO₂. 50 μl of GM-CSF diluted to the EC₈₀ value inassay medium (or 0.4 ng/ml for some batches of GM-CSF) was then added toeach well and the plates were incubated for 16 hours at 37° C. in 5% CO₂in a humidified chamber. This represents a final concentration of 7 pMGM-CSF. In order to measure the proliferation of the cells, 20 μl of ³H-thymidine diluted to 5.0 μCi/ml in assay medium was added to each wellof the plate and the plates were incubated for 4 hours±30 mins at 37° C.in 5% CO₂. Cells were then harvested onto 96 well GF/C UNIFILTER™ platesusing a plate harvester and washed. After adding 50 μl MicroScint 20™ toeach well of the filter plate, the plates were sealed and counted on aTOPCOUNT® plate reader.

Human Granulocyte Shape Change Assay

Human buffy coats (human blood pack from the Blood Transfusion service)were mixed in an equal volume of 3% Dextran T-500 in 0.9% NaCl. Themixture was then incubated in an upright position until an interface hadformed. The upper layer was harvested and layered on top of aHISTOPAQUE® 1.077 density gradient which was then centrifuged at 400 gfor 40 minutes and allowed to stop without braking. The upper layers ofthis gradient were removed leaving the granulocyte pellet. Any remainingred blood cells in the pellet were lysed by resuspending the cells in 20ml of ice cold water for 30s followed by the immediate addition of icecold 1.8% sodium chloride. Cells were then repelleted at 1200 rpm andresuspended in assay medium (RPMI1640, 10% FBS, 100u/ml Penicillin, 100μg/ml streptomycin, 25 mM HEPES) at 1×10⁶/ml. 100 μl of cells was thenadded to each well of a 96 well flat bottomed tissue culture plate. Testsamples were prepared by sterile filtering the stock samples anddiluting, as appropriate, in assay medium.

For lead isolation, 50 μl of test sample was then added to the cells andthe plates were incubated for 45-60 mins at 37° C. in 5% CO₂. Thisrepresents a final concentration of 7 pM GM-CSF. This was followed bythe addition of 5001 of GM-CSF diluted to 0.4 ng/ml in assay medium toeach well and a 4 hour incubation at 37° C. in 5% CO₂ in a humidifiedchamber.

For lead optimisation, filtered IgG4s diluted in assay medium were mixedwith an equal volume of GM-CSF at 0.4 ng/ml in assay medium. Thisrepresents a final concentration of 7 pM GM-CSF. 100 μl ofantibody/GM-CSF mix was then added to each well. This was followed by a3 hour incubation at 37° C. in 5% CO₂ in a humidified chamber.

Cold formaldehyde was added to a final concentration of 1.25% and cellswere fixed overnight at 4° C. 2000-5000 events per well were analysed byflow cytometry. The geometric mean of the forward scatter (FSC) for eachsample was then derived using CellQuest. Cells were gated to excludeirrelevant populations (e.g. dead cells/debris) when calculating thegeometric mean.

Cynomolgus Granulocyte Shape Change Assay

Antibodies were assessed in an assay measuring the shape change ofcynomolgus granulocytes following stimulation with GM-CSF. Granulocyteswere purified from whole cynomolgus blood and the assay was carried outessentially as described for the human granulocyte shape change assay.

Binding Affinity Data Using Biosensor Analysis

The BIACORE® 2000 System (Pharmacia Biosensor) was used to assess thekinetic parameters of the interaction between scFvs and IgG4s with therecombinant receptors. The Biosensor uses the optical effects of surfaceplasmon resonance to study changes in surface concentration resultingfrom the interaction of an analyte molecule with a ligand molecule thatis covalently attached to a dextran matrix. Typically the analytespecies in free solution is passed over the coupled ligand and anybinding is detected as an increase in local SPR signal. This is followedby a period of washing, during which dissociation of the analyte speciesis seen as a decrease in SPR signal, after which any remaining analyteis stripped from the ligand and the procedure repeated at severaldifferent analyte concentrations. A series of controls are usuallyemployed during an experiment to ensure that neither the absolutebinding capacity nor kinetic profile of the coupled ligand changesignificantly. A proprietary HEPES buffer saline (HBS-EP) is typicallyused as the main diluent of analyte samples and dissociation phasesolvent. The experimental data is recorded in resonance units (directlycorresponding to the SPR signal) with respect to time. The resonanceunits are directly proportional to the size and quantity of analytebound. The BIAevaluation software package can then be used assign rateconstant to the dissociation phase (dissociation rate units s⁻¹) andassociation phase (association rate units M⁻¹ s⁻¹). These figures thenallow calculation of the Association and Dissociation AffinityConstants.

The affinity of IgG4 was estimated using a single assay in which theIgG4 was non-covalently captured by amine protein A surface. A series ofdilutions of recombinant purification-tagged GM-CSF receptorextracellular domain, from 100 to 6.25 nM were then sequentially passedover the IgG4. The molarity of the receptor was calculated using theconcentration (Bradford) and the predicted non post-translationallymodified mature polypeptide mass (39.7 kDa). Each of the two separatesets of data were analysed in identical formats. Reference cellcorrected data was subject to fitting using the 1:1 langmuir model setfor simultaneous global calculation of the association and dissociationrates, with the Rmax value set to global. The level of IgG4 capturedduring each cycle was assessed to ensure that the quantity capturedremained stable during the entire experiment. Additionally, thedissociation rate of the IgG4 was assessed to determine if a correctionfor baseline drift was required. However, both the protein Ainteractions proved to be sufficiently reproducible and stable. Thevalidity of the data was constrained by the calculated chi2 and T value(parameter value/offset), which had to be <2 and >100 respectively.

Production of purification-tagged GM-CSFRα extracellular domain: ApEFBOS expression vector [106] incorporating a sequence encoding humanGM-CSF receptor α extracellular domain (SEQ ID NO: 205, representingamino acids 1 to 298 of the mature GM-CSF R) with a murine IL-3 signalsequence and incorporating an N-terminal purification tag was used toproduce recombinant N-terminal tagged GM-CSF receptor extracellulardomain (ECD) polypeptide. The tagged ECD polypeptide was expressed inCHO cells using the pEFBOS vector using standard procedures. Thispolypeptide may also be referred to as purified GM-CSFRα extracellulardomain, or as the soluble extracellular domain of GM-CSFRα.

Any suitable purification tag may be used e.g. Flag peptide (DYKDDDE—SEQID NO: 204), Fc, biotin or his tag. Purification can be conducted usingany appropriate technique, e.g. a Flag-tagged ECD polypeptide (SEQ IDNO: 203) may be purified on an M2 affinity chromatography column andeluted with FLAG peptide.

Monocyte TNFα Release Assay

Purification of Monocytes (Monocyte Isolation Kit—MiltenyiBiotec-130-053-301):

Human buffy coats (human blood pack from the Blood Transfusion service)were layered on top of a HISTOPAQUE® 1.077 density gradient (Sigma, CatNo. 1077-1) and cells were centrifuged at 400×g for 40 minutes. No brakewas applied when stopping the centrifuge. PBMC cells were then harvestedfrom the interface. Cells were washed in PBS and pelleted at 300×g for10 mins before the remaining red blood cells were lysed by resuspensionin 20 ml of ice cold water for 15 s followed by the immediate additionof ice cold 1.8% NaCl. Cells were then pelleted at 1200 rpm for 5 minsand resuspended in 600 μl of MACS buffer (PBS, 2 mM EDTA). 200 μl of Fcblocking reagent provided with the kit was added to the cells and mixedbefore adding 200 μl of Hapten-antibody cocktail (also provided with thekit) and mixing. Cells were then incubated at 4° C. for 15 mins beforewashing twice in 50 ml of MACS buffer. The cell pellet was resuspendedin 600 μl of MACS buffer before adding 200 μl of Fc blocking reagent andmixing followed by 200 μl of MACS anti-hapten microbeads and mixing. Thecells were incubated for 45 mins at 4° C. before washing in 50 ml MACSbuffer and resuspending in 500 μl of MACS buffer. A single column(Miltenyi Biotec 130-042-401) was prepared by washing through with 3 mlof MACS buffer before the cell suspension was applied to the column. Theeffluent was collected as the enriched monocyte fraction. The column waswashed with 2×3 ml MACS buffer and the effluent was collected. Monocytepurity was checked by staining with anti-CD14-PE using standard flowcytometry methods. Cells were finally resuspended at 4×10⁶/ml in assaymedium (RPMI 1640, 10% FCS, 100 U/ml penicillin, 100 μg/mlstreptomycin).

Stimulation of Monocytes:

50 μl of cells were added to each well of a COSTAR® 96 wellflat-bottomed tissue culture plate. 25 μl of 150 μg/ml rhIFNγ (R&Dsystems) was added to all wells. Filtered IgG4s diluted in assay mediumwere mixed with an equal volume of GM-CSF at 56 ng/ml (4 nM) in assaymedium. This represents a final concentration of 1 nM GM-CSF. 75 μl ofantibody/GM-CSF mix was then added to each well. Controls were wellswith GM-CSF only or with no GM-CSF and no antibody. Plates were thenincubated for 18 hours at 37° C. with 5% CO₂ in a humidified chamber.The supernatant was then harvested to test for TNF-α levels by ELISA.

TNFα ELISA (R&D Systems ELISA Development System DY210): FLUORONUNC™Immunosorb ELISA plates were coated overnight at room temperature with100 μl of capture antibody at 4 μg/ml in PBS. Plates were then washedthree times with PBS/0.1% Tween and blocked with 300 μl/well of 3%Marvel in PBS for 1 hour at room temperature. Plates were washed 3 timeswith PBS/0.1% Tween. 100 μl of the supernatant from the assay plates wastransferred to the ELISA plate and a titration of TNF-α diluted in assaymedium was added to the control wells. Plates were incubated at roomtemperature for 2 hours before washing 4-5 times with PBS/0.1% Tween.100 μl of detection antibody diluted to 300 ng/ml in 1% Marvel/PBS wasadded to each well of the plate and the plates were incubated for afurther 2 hours at room temperature before washing 4-5 times withPBS/0.1% Tween. Streptavidin-Europium (PerkinElmer 1244-360) was diluted1:1000 in DELFIA® assay buffer (PerkinElmer 4002-0010) and added at 100μl/well before incubating for 45 mins at room temperature. Plates werethen washed 7 times in DELFIA® wash buffer before the addition of 100μl/well of enhancement solution (PerkinElmer 4001-0010) and reading at615 nm on a platereader.

Granulocyte Survival Assay

Cells were purified from human buffy coats as described for theneutrophil activation assay (shape change assay) washed in assay medium(RPMI-1640 GLUTAMAX™, 10% FBS, 100 U/ml Penicillin, 100 μg/mlstreptomycin) and resuspended at 1×10⁶/ml in assay medium. 100 μl ofcells were added to each well of a COSTAR® 96 well flat-bottomed tissueculture plate. Filtered stocks of antibody were diluted in assay mediumand mixed with an equal volume of GM-CSF at 0.4 ng/ml. This represents afinal concentration of 7 pM GM-CSF. Control wells contained media aloneor GM-CSF alone. 100 μl of the test sample/GM-CSF mix was then added toeach well on the plate and the cells were incubated for 68 hours at 37°C./5% CO₂ in a humidified chamber. 20 μl of ALAMARBLUE® was added toeach well and the plates were incubated for a further 24 hours at 37°C./5% CO₂ in a humidified chamber. Plates were then read at 560 nm and590 nm on a platereader.

pA₂ Analysis of anti-GM-CSFRα Antibodies in the TF-1 Proliferation Assayand in the Human and Cynomolgus Granulocyte Shape Change Assays

The main pharmacological tool to quantify the affinity of a competitiveantagonist is Schild analysis. Using this approach a system-independentmeans of estimating the antagonist affinity in a functional assay maybedetermined. The method is based on the concept that the antagonistconcentration and its affinity determines the antagonism of the agonistresponse. Because the antagonism can be quantified and the concentrationof the antagonist is known, the affinity of the antagonist can bedetermined. This antagonism is quantified by measuring the ratio ofequiactive concentrations of agonists, measured in the presence andabsence of the antagonist, referred to as dose ratios (DR).

Dose ratios may be calculated by taking the ratio of the EC50 of agonist(typically GM-CSF) in the absence of the binding member to the EC50 ofthe agonist in the presence of a single concentration of binding member.The dose ratios, expressed as log(DR-1) may then be used in a linearregression on log [binding member] to produce a Schild regression. Thus,for every concentration of binding member there will be a correspondingDR value; these are plotted as the regression of log(DR-1) upon log[binding member]. If the antagonism is competitive, there will be alinear relationship between log (DR-1) and log [binding member]according to the Schild equation wherein the equation is as followsLog(DR-1)=log [A]−log K _(A)

Under these circumstances, a value of zero for the ordinate will give anintercept of the x-axis where log [a]=log K_(A). Therefore theconcentration of binding member that produces a log (DR-1)=0 will beequal to the log K_(A), the equilibrium dissociation constant of thebinding member—receptor complex. This is a system independentquantification of the binding member affinity that should be accuratefor every cellular system containing the receptor.

Because the K_(A) values are obtained from a logarithmic plot, they arelog normally distributed. The negative logarithm of this particularconcentration is referred to empirically as pA2, the concentration ofantagonist that produces a twofold shift of the agonist dose responsecurve. The antagonist potency can be quantified by calculating pA2 froma single concentration of antagonist producing a single value for thedose ratio from the equation, whereinpA ₂=log(DR-1)−log [a][a]=molar concentration of antagonist that makes it necessary to doublethe agonist concentration to elicit the original submaximal response.DR=the dose ratio is quantified by measuring the ratio of equiactiveconcentrations of agonist measured in the presence and absence of theantagonist.pA₂ may be calculated from dose-response assay data.Inhibition of In Vitro GM-CSF Mediated Differentiation of Blood CellProgenitors in Colony Formation Assay

Peripheral blood mononuclear cells enriched for haemopoietic progenitorcells were obtained from donors who had undergone progenitor cellmobilisation and apheresis as part of their standard clinicalmanagement. Samples were de-identified and cells were not cryopreservedprior to use. 5×10⁴ mononuclear cells were cultured in semi-solid agar[107] in the presence of human GM-CSF at a final concentration of 10ng/ml. Test affinity matured human mAbs, and the known murine anibody2B7, were added to agar cultures at a final concentration of 10, 5, 1,0.5, 0.1 or 0.05 μg/ml. The parent human mAb 28G5 and an isotype matchednegative control human mAb, CAT001, were assessed at a singleconcentration of 10 μg/ml. For control purposes mAbs were also assessedfor their ability to block colony formation stimulated by a combinationof SCF, IL-3 and G-CSF (Croker et al., 2004) and for their impact oncolony formation in the absence of cytokines. Colony formation(aggregates of >40 cells) was assessed after 14 days incubation at 37°C. with 10% CO₂ in air. Colonies were fixed with glutaraldehyde andcounted using a dissection microscope at a magnification of 35×.

Inhibition of GM-CSF Biological Activity In Vivo in Human GM-CSFRαβTransgenic Mice

Transgenic (Tg) mice expressing both the α and the β chains of the humanGM-CSFR under the control of an MHC class I promoter have been generatedand in vivo spleen and blood cell responses to administration ofhuGM-CSF have been described [108]. For in vivo analysis of huGM-CSFRαspecific mAb antagonist activity, transplantation of bone marrow fromthe Tg mice into wildtype mice can be used to generate chimeric animalssuch that transgenic huGM-CSFRαβ expression is limited to bone marrowderived haemopoietic cells and thus more closely resembles theexpression profile of the endogenous receptor. In these huGM-CSFRαβ Tgchimeric mice the administration of huGM-CSF leads to an increase inspleen weight and the marginalisation of circulating blood monocytes.

Generation of Tg Chimeric Mice:

Femurs and tibiae from donor Tg mice were removed and the bone marrowflushed out with sterile PBS plus 3% fetal calf serum (FCS). The bonemarrow plugs were then drawn up through a 23G needle to obtain a singlecell suspension, then cells washed once with cold PBS+3% FCS and passedthrough a stainless steel mesh. Red cells were then removed by lysis in0.168 M ammonium chloride buffer, after which cells were washed twicemore with phosphate buffered saline (PBS)+3% FCS before again beingpassed through a stainless steel mesh. To further remove dead cells andcell debris the suspension was centrifuged through an FCS cushion.Viable cells are recovered in the pellet, washed once with PBS andresuspended in PBS at 2.5×10⁷/ml. 5 to 8 week old recipient C57/BL6 micewere lethally irradiated with 2 doses of 550 Rad, 3 hours apart.Recipient mice were injected intravenously (i.v) with 0.2 ml cellsuspension (ie. 5×10⁶ cells/mouse) and subsequently housed in hoodedboxes with 0.02 M neomycin in their drinking water for 3 weeks.Reconstitution was assessed after 6 weeks by FACS analysis of peripheralblood using mAbs specific for the huGMCSFRα and β chains.

GM-CSF treatment and subsequent analysis of Tg chimeric mice: Tgchimeric mice were treated twice daily via the subcutaneous (s.c) routewith 500 ng of huGM-CSF for 4 days. For analysis of antibody antagonistactivity groups of 5 mice were administered selected doses of mAb (seebelow) via the intraperitoneal (i.p) route 1 day prior to initiation ofGM-CSF treatment. At day 5, 0.2 ml of blood was sampled for analysis ofcirculating leukocyte populations, in particular blood monocytes, usingan ADVIA™ Hematology System (Bayer Diagnostics). Animals were thensacrificed and spleens removed for weight measurement.

Inhibition of endogenously expressed human TNFa and IL-6 from humanperipheral blood mononuclear cells

Human buffy coats (human blood pack from the Blood Transfusion service)were layered on top of a HISTOPAQUE® 1.077 density gradient (Sigma, CatNo. 1077-1) and cells were centrifuged at 400×g for 40 minutes. No brakewas applied when stopping the centrifuge. PBMC cells were then harvestedfrom the interface. Cells were washed in PBS and pelleted at 300×g for10 mins before the remaining red blood cells were lysed by resuspensionin 20 ml of ice cold water for 15 s followed by the immediate additionof ice cold 1.6% NaCl. Cells were then pelleted at 1200 rpm for 5 minsand resuspended in 10 ml of 10% FBS/RPMI and 1% penicillin streptomycin.Cells were then diluted to 5×10⁶/ml. 110 μl of cells were dispensed perwell (5.5×10⁵/well) and cells allowed to settle for 1 hr at 37° C., 5%CO₂. The following reagents were added as single final concentrationcontrols; PHA (5 μg/ml), LPS (25 μg/ml), GM-CSF (10 ng/ml) and isotypecontrol (50 μg/ml). Antibody 6 was added to a final startingconcentration of 50 μg/ml with a fivefold dilution series. Plates werethen incubated for 72 hrs at 37° C., 5% CO₂. Supernatants were harvestedafter 72 hrs and the levels of TNFa and IL-6 were calculated using thefollowing R&D ELISA kits (hTNF-a R&D Duoset ELISA development systemDY210 and hIL-6 R&D Duoset ELISA development system DY206). ELISA wereperformed according to suppliers recommendations.

TABLE 1 Inhibition of GM-CSF induced proliferation of TF-1 cells by IgG4non-germlined antibodies isolated from optimisation of 28G5. IC50 ± SEMIgG4 (pM) 2B7  1575 ± 490.5 antibody 1  5.3 ± 0.33 antibody 2 15.0 ±4.71 antibody 4 48.0 ± 8.33 antibody 5  9.3 ± 5.39 antibody 6  0.97 ±0.033 antibody 7 93.8 ± 24.6 antibody 8 34.5 ± 2.63 antibody 9 40.8 ±7.15 antibody 10 55.3 ± 3.73 antibody 11 9.0 ± 1.0 antibody 12 246.3 ±19.8  antibody 13 1106.0 ± 174.9  antibody 14 16.3 ± 4.9  antibody 15163.8 ± 7.3  antibody 16 12.8 ± 3.3  antibody 17 14.3 ± 2.8  antibody 1813.3 ± 3.4  antibody 19 23.8 ± 4.3  antibody 20 9.8 ± 2.8 Proliferationof TF-1 cells was induced with a single concentration of GM-CSF in thepresence of increasing concentrations of IgG4 antibodies. Theincorporation of tritiated thymidine was measured and IC50 values forthe antibodies were calculated. Data are representative of n ≧ 3. SEM(standard error of the mean) is shown.

TABLE 2 Kinetic analysis of anti-GM-CSFRα IgG4 non-germlined antibodiesisolated during optimisation of 28G5. IgG4 KD (nM) antibody 1 0.264antibody 2 0.376 antibody 4 4.07 antibody 5 0.847 antibody 6 0.139antibody 7 3.93 antibody 8 0.552 antibody 10 1.50 antibody 12 3.02antibody 14 0.502 antibody 15 1.03 antibody 16 1.14 antibody 17 0.193antibody 19 0.388 antibody 20 0.127 IgG4 antibodies were immobilised tothe surface of a protein-A coated chip and a series ofpurification-tagged GM-CSF Rα ECD dilutions were passed over the IgG4.Data was subject to fitting using the Langmuir 1:1 simultaneous k_(a)k_(d) with allowance for mass transport.

Data for antibodies 9 and 11 were biphasic.

TABLE 3 Inhibition of GM-CSF induced shape change of human granulocytesby IgG4 non-germlined antibodies isolated during optimisation of 28G5.IC50 ± SD IgG4 (pM) 2B7 477 ± 491 antibody 1 12.6 ± 8.0  antibody 2 20.7± 11.0 antibody 5 30.0 antibody 6 13.3 ± 11.8 antibody 9 44.0 antibody10 62.0 antibody 11 90.0 antibody 16 16.0 antibody 20  7.8 Humangranulocytes were treated with a single concentration of GM-CSF in thepresence of increasing concentrations of IgG4 antibody. The change inshape of the granulocytes was measured using flow cytometry and IC50values for the antibodies were calculated.

TABLE 4 Inhibition of GM-CSF induced release of TNFα from monocytes.IC50 ± SD IgG4 (pM) antibody 1 78.8 ± 54.6 antibody 2 103.3 ± 63.1 antibody 5 67.0 antibody 6 43.0 ± 19.7 antibody 9 74.0 antibody 10139.0  Human monocytes were treated with a single concentration ofGM-CSF in the presence of increasing concentrations of IgG4non-germlined antibody. The release of TNFα was measured by ELISA andthe IC50 values for the antibodies were calculated.

TABLE 5 KABAT RESIDUE 28G5 LEAD Percentage occurrence of residues H95 VV A N L V 1.250 26.875  1.250 70.625  H97 S S S 100.00   H99 S S P S T HF W 1.250 70.625  0.625 0.625 26.250  0.625 H100B A T A P S T H V63.125  2.500 2.500 28.75  0.625 2.500 L90 S T S T M 90.000  9.375 0.625L92 D E S T P Q E M 2.500 0.625 91.875  1.875 2.500 0.625 L95A S S G P ST K D Q E R 9.375 1.250 45.000  3.125 6.250 6.875 5.625 4.375 3.125 H KI L M V F Y 4.375 2.500 1.250 1.875 0.625 0.625 0.625 3.125 L96 S S A PS T I L M V 1.250 26.250  43.750  1.250 17.500  0.625 1.250 8.125Key to Sequence Listing

The Sequence Listing is submitted as an ASCII text file entitled“Sequences” created on Feb. 10, 2011, ˜100 KB, which is incorporated byreference herein. In the appended sequence listing, nucleic acid andamino acid (“PRT”) sequences are listed for 20 antibody clones,comprising the parent clone and the 19 clones from the optimised panel.Antibodies are numbered Ab1 to Ab20. The parent clone is antibody 3,represented by SEQ ID NOS: 21, 22, 3, 4, 25, 26, 17, 8, 9, and 20 andSEQ ID NOS: 209-211.

The following list identifies by number the SEQ ID NOS in whichsequences of the indicated molecules are shown:

(nt=nucleotide sequence; aa=amino acid sequence

1 Antibody 01 VH nt 2 Antibody 01 VH aa 3 Antibody 01 VH CDR1 aa 4Antibody 01 VH CDR2 aa 5 Antibody 01 VH CDR3 aa 6 Antibody 01 VL nt 7Antibody 01 VL aa 8 Antibody 01 VL CDR1 aa 9 Antibody 01 VL CDR2 aa 10Antibody 01 VL CDR3 aa 11 Antibody 02 VH nt 12 Antibody 02 VH aa 3Antibody 02 VH CDR1 aa 4 Antibody 02 VH CDR2 aa 15 Antibody 02 VH CDR3aa 16 Antibody 02 VL nt 17 Antibody 02 VL aa 8 Antibody 02 VL CDR1 aa 9Antibody 02 VL CDR2 aa 20 Antibody 02 VL CDR3 aa 21 Antibody 03 VH nt 22Antibody 03 VH aa 3 Antibody 03 VH CDR1 aa 4 Antibody 03 VH CDR2 aa 25Antibody 03 VH CDR3 aa 26 Antibody 03 VL nt 17 Antibody 03 VL aa 8Antibody 03 VL CDR1 aa 9 Antibody 03 VL CDR2 aa 20 Antibody 03 VL CDR3aa 31 Antibody 04 VH nt 32 Antibody 04 VH aa 3 Antibody 04 VH CDR1 aa 4Antibody 04 VH CDR2 aa 35 Antibody 04 VH CDR3 aa 36 Antibody 04 VL nt 17Antibody 04 VL aa 8 Antibody 04 VL CDR1 aa 9 Antibody 04 VL CDR2 aa 20Antibody 04 VL CDR3 aa 41 Antibody 05 VH nt 42 Antibody 05 VH aa 3Antibody 05 VH CDR1 aa 4 Antibody 05 VH CDR2 aa 45 Antibody 05 VH CDR3aa 36 Antibody 05 VL nt 17 Antibody 05 VL aa 8 Antibody 05 VL CDR1 aa 9Antibody 05 VL CDR2 aa 20 Antibody 05 VL CDR3 aa 51 Antibody 06 VH nt 52Antibody 06 VH aa 3 Antibody 06 VH CDR1 aa 4 Antibody 06 VH CDR2 aa 55Antibody 06 VH CDR3 aa 56 Antibody 06 VL nt 57 Antibody 06 VL aa 8Antibody 06 VL CDR1 aa 9 Antibody 06 VL CDR2 aa 60 Antibody 06 VL CDR3aa 61 Antibody 07 VH nt 62 Antibody 07 VH aa 3 Antibody 07 VH CDR1 aa 4Antibody 07 VH CDR2 aa 65 Antibody 07 VH CDR3 aa 36 Antibody 07 VL nt 17Antibody 07 VL aa 8 Antibody 07 VL CDR1 aa 9 Antibody 07 VL CDR2 aa 20Antibody 07 VL CDR3 aa 71 Antibody 08 VH nt 72 Antibody 08 VH aa 3Antibody 08 VH CDR1 aa 4 Antibody 08 VH CDR2 aa 75 Antibody 08 VH CDR3aa 36 Antibody 08 VL nt 17 Antibody 08 VL aa 8 Antibody 08 VL CDR1 aa 9Antibody 08 VL CDR2 aa 20 Antibody 08 VL CDR3 aa 81 Antibody 09 VH nt 82Antibody 09 VH aa 3 Antibody 09 VH CDR1 aa 4 Antibody 09 VH CDR2 aa 85Antibody 09 VH CDR3 aa 36 Antibody 09 VL nt 17 Antibody 09 VL aa 8Antibody 09 VL CDR1 aa 9 Antibody 09 VL CDR2 aa 20 Antibody 09 VL CDR3aa 91 Antibody 10 VH nt 92 Antibody 10 VH aa 3 Antibody 10 VH CDR1 aa 4Antibody 10 VH CDR2 aa 95 Antibody 10 VH CDR3 aa 36 Antibody 10 VL nt 17Antibody 10 VL aa 8 Antibody 10 VL CDR1 aa 9 Antibody 10 VL CDR2 aa 20Antibody 10 VL CDR3 aa 101 Antibody 11 VH nt 102 Antibody 11 VH aa 3Antibody 11 VH CDR1 aa 4 Antibody 11 VH CDR2 aa 105 Antibody 11 VH CDR3aa 106 Antibody 11 VL nt 107 Antibody 11 VL aa 8 Antibody 11 VL CDR1 aa9 Antibody 11 VL CDR2 aa 20 Antibody 11 VL CDR3 aa 21 Antibody 12 VH nt22 Antibody 12 VH aa 3 Antibody 12 VH CDR1 aa 4 Antibody 12 VH CDR2 aa115 Antibody 12 VH CDR3 aa 116 Antibody 12 VL nt 117 Antibody 12 VL aa 8Antibody 12 VL CDR1 aa 9 Antibody 12 VL CDR2 aa 120 Antibody 12 VL CDR3aa 21 Antibody 13 VH nt 22 Antibody 13 VH aa 3 Antibody 13 VH CDR1 aa 4Antibody 13 VH CDR2 aa 125 Antibody 13 VH CDR3 aa 126 Antibody 13 VL nt127 Antibody 13 VL aa 8 Antibody 13 VL CDR1 aa 9 Antibody 13 VL CDR2 aa130 Antibody 13 VL CDR3 aa 131 Antibody 14 VH nt 132 Antibody 14 VH aa 3Antibody 14 VH CDR1 aa 4 Antibody 14 VH CDR2 aa 135 Antibody 14 VH CDR3aa 136 Antibody 14 VL nt 137 Antibody 14 VL aa 8 Antibody 14 VL CDR1 aa9 Antibody 14 VL CDR2 aa 140 Antibody 14 VL CDR3 aa 141 Antibody 15 VHnt 142 Antibody 15 VH aa 3 Antibody 15 VH CDR1 aa 4 Antibody 15 VH CDR2aa 145 Antibody 15 VH CDR3 aa 146 Antibody 15 VL nt 147 Antibody 15 VLaa 8 Antibody 15 VL CDR1 aa 9 Antibody 15 VL CDR2 aa 150 Antibody 15 VLCDR3 aa 151 Antibody 16 VH nt 152 Antibody 16 VH aa 3 Antibody 16 VHCDR1 aa 4 Antibody 16 VH CDR2 aa 155 Antibody 16 VH CDR3 aa 156 Antibody16 VL nt 157 Antibody 16 VL aa 8 Antibody 16 VL CDR1 aa 9 Antibody 16 VLCDR2 aa 160 Antibody 16 VL CDR3 aa 161 Antibody 17 VH nt 152 Antibody 17VH aa 3 Antibody 17 VH CDR1 aa 4 Antibody 17 VH CDR2 aa 165 Antibody 17VH CDR3 aa 166 Antibody 17 VL nt 167 Antibody 17 VL aa 8 Antibody 17 VLCDR1 aa 9 Antibody 17 VL CDR2 aa 170 Antibody 17 VL CDR3 aa 171 Antibody18 VH nt 172 Antibody 18 VH aa 173 Antibody 18 VH CDR1 aa 4 Antibody 18VH CDR2 aa 175 Antibody 18 VH CDR3 aa 176 Antibody 18 VL nt 177 Antibody18 VL aa 8 Antibody 18 VL CDR1 aa 9 Antibody 18 VL CDR2 aa 180 Antibody18 VL CDR3 aa 181 Antibody 19 VH nt 182 Antibody 19 VH aa 3 Antibody 19VH CDR1 aa 4 Antibody 19 VH CDR2 aa 185 Antibody 19 VH CDR3 aa 186Antibody 19 VL nt 187 Antibody 19 VL aa 8 Antibody 19 VL CDR1 aa 9Antibody 19 VL CDR2 aa 190 Antibody 19 VL CDR3 aa 191 Antibody 20 VH nt192 Antibody 20 VH aa 3 Antibody 20 VH CDR1 aa 4 Antibody 20 VH CDR2 aa195 Antibody 20 VH CDR3 aa 196 Antibody 20 VL nt 197 Antibody 20 VL aa 8Antibody 20 VL CDR1 aa 9 Antibody 20 VL CDR2 aa 200 Antibody 20 VL CDR3aa 201 GM-CSFRα linear residue sequence 202 Full length amino acidsequence of human GM-CSFRα 203 FLAG-tagged human GM-CSFRα extracellulardomain 204 FLAG peptide 205 Amino acid sequence of human GM-CSFRαextracellular domain 206 Mature GM-CSFRα 207 Antibody 1 VL nt 208Antibody 1 VL aa 209 Antibody 2 VL nt 210 Antibody 2 VL aa 211 Antibody3 VL nt 210 Antibody 3 VL aa 209 Antibody 4 VL nt 210 Antibody 4 VL aa209 Antibody 5 VL nt 210 Antibody 5 VL aa 217 Antibody 6 VL nt 218Antibody 6 VL aa 209 Antibody 7 VL nt 210 Antibody 7 VL aa 209 Antibody8 VL nt 210 Antibody 8 VL aa 209 Antibody 9 VL nt 210 Antibody 9 VL aa209 Antibody 10 VL nt 210 Antibody 10 VL aa 227 Antibody 11 VL nt 228Antibody 11 VL aa 229 Antibody 12 VL nt 230 Antibody 12 VL aa 231Antibody 13 VL nt 232 Antibody 13 VL aa 233 Antibody 14 VL nt 234Antibody 14 VL aa 235 Antibody 15 VL nt 236 Antibody 15 VL aa 237Antibody 16 VL nt 238 Antibody 16 VL aa 239 Antibody 17 VL nt 240Antibody 17 VL aa 241 Antibody 18 VL nt 242 Antibody 18 VL aa 243Antibody 19 VL nt 244 Antibody 19 VL aa 245 Antibody 20 VL nt 246Antibody 20 VL aa 247 Antibody 6 VH nt 248 Antibody 6 VH aa 249 Antibody6 VL nt 250 Antibody 6 VL aa 251 VH FR1 aa 252 VH FR2 aa 253 VH FR3 aa254 VH FR4 aa 255 VL FR1 aa 256 VL FR2 aa 257 VL FR3 aa 258 VL FR4 aa

The VL domain nucleotide sequences of antibodies 1 to 20 do not includethe gcg codon shown at the 3′ end in SEQ ID NOS: 6, 16, 26, 36, 56, 106,116, 126, 136, 146, 156, 166, 176, 186 and 196. Correspondingly, the VLdomain amino acid sequences do not include the C-terminal Ala residue(residue 113) in SEQ ID NOS: 7, 17, 57, 107, 117, 127, 137, 147, 157,167, 177, 187 and 197. The Ala113 residue and corresponding gcg codonwere not expressed in Antibodies 1 to 20. A comparison of the writtensequences with germline gene segments, especially JL2, indicates thatthe Ala residue and corresponding gag codon do not form part of the VLdomain.

The Gly residue at position 112 was present in the expressed scFv andIgG sequences. However, this residue is not present in human germline jsegment sequences that form the framework 4 region of the VL domain,e.g. JL2. The Gly residue is not considered a part of the VL domain.

To express the light chain of the IgG, a nucleotide sequence encodingthe antibody light chain was provided, comprising a first exon encodingthe VL domain, a second exon encoding the CL domain, and an intronseparating the first exon and the second exon. Under normalcircumstances, the intron is spliced out by cellular mRNA processingmachinery, joining the 3′ end of the first exon to the 5′ end of thesecond exon. Thus, when DNA having the said nucleotide sequence wasexpressed as RNA, the first and second exons were spliced together.Translation of the spliced RNA produces a polypeptide comprising the VLand the CL domain. After splicing, the Gly at position 112 is encoded bythe last base (g) of the VL domain framework 4 sequence and the firsttwo bases (gt) of the CL domain.

The VL domain sequences of Antibodies 1 to 20 are SEQ ID NOS: 186 to 246as indicated above. The VL domain nucleotide sequences end with cta asthe final codon, and Leu is the final amino acid residue in thecorresponding VL domain amino acid sequences.

Non-germlined VH and VL domain sequences of Antibody 6 are shown in SEQID NOS: 247-250, in addition to the germlined VH and VL domain sequencesshown in SEQ ID NOS: 51, 52, 56, 57, 216 and 217.

REFERENCES

All documents mentioned anywhere in this disclosure are incorporatedherein by reference.

-   1 Haman et al., Journal of Biological Chemistry 274(48):34155-34163    1999-   2 Nicola N A; Wycherley A; Boyd A W; Layton J E; Cary D; Metcalf D    Blood, 82(6) p1724-31 (1993)-   3 Plückthun, A. Bio/Technology 9: 545-551 (1991)-   4 Chadd H E and Chamow S M (2001) Current Opinion in Biotechnology    12: 188-194-   5 Andersen D C and Krummen L (2002) Current Opinion in Biotechnology    13: 117-   6 Larrick J W and Thomas D W (2001) Current Opinion in Biotechnology    12:411-418-   7 Sambrook and Russell, Molecular Cloning: a Laboratory Manual: 3rd    edition, 2001, Cold Spring Harbor Laboratory Press-   8 Ausubel et al. eds., Short Protocols in Molecular Biology: A    Compendium of Methods from Current Protocols in Molecular Biology,    John Wiley & Sons, 4^(th) edition 1999-   9 Wold, et al. Multivariate data analysis in chemistry.    Chemometrics—Mathematics and Statistics in Chemistry (Ed.: B.    Kowalski), D. Reidel Publishing Company, Dordrecht, Holland, 1984    (ISBN 90-277-1846-6)-   10 Norman et al. Applied Regression Analysis. Wiley-Interscience;    3^(rd) edition (April 1998) ISBN: 0471170828-   11 Kandel, Abraham & Backer, Eric. Computer-Assisted Reasoning in    Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN:    0133418847-   12 Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's    Perspective (Oxford Statistical Science Series, No 22 (Paper)).    Oxford University Press; (December 2000), ISBN: 0198507089-   13 Witten, Ian H. & Frank, Eibe. Data Mining: Practical Machine    Learning Tools and Techniques with Java Implementations. Morgan    Kaufmann; (Oct. 11, 1999), ISBN: 1558605525-   14 Denison David G. T. (Editor), Christopher C. Holmes, Bani K.-   15 Ghose, Arup K. & Viswanadhan, Vellarkad N. Combinatorial Library    Design and Evaluation Principles, Software, Tools, and Applications    in Drug Discovery. ISBN: 0-8247-0487-8-   16 Chothia C. et al. Journal Molecular Biology (1992) 227,-   17 Al-Lazikani, et al. Journal Molecular Biology (1997) 273(4),    927-948-   18 Chothia, et al. Science, 223, 755-758 (1986)-   19 Whitelegg, N. R. u. and Rees, A. R (2000). Prot. Eng., 12,    815-824-   20 Guex, N. and Peitsch, M. C. Electrophoresis (1997) 18, 2714-2723-   21 Voet & Voet, Biochemistry, 2nd Edition, (Wiley) 1995.-   22 Marks et al Bio/Technology, 1992, 10:779-783-   23 Kay, B. K., Winter, J., and McCafferty, J. (1996) Phage Display    of Peptides and Proteins: A Laboratory Manual, San Diego: Academic    Press.-   24 Stemmer, Nature, 1994, 370:389-391-   25 Gram et al., 1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580-   26 Barbas et al., 1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813-   27 Schier et al., 1996, J. Mol. Biol. 263:551-567-   28 Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664-   29 Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates and    Radiopharmaceuticals 4: 915-922-   30 Robinson, J. R. ed., Sustained and Controlled Release Drug    Delivery Systems, Marcel Dekker, Inc., New York, 1978-   31 Ritz, S. A., M. J. Cundall, et al. (2002). Am J Respir Cell Mol    Biol 27(4): 428-35.-   32 Ritz, S. A., M. R. Stampfli, et al. (2002). Trends Immunol 23(8):    396-402. 33 WOOLLEY, K. L., et al. (1995). Am J Respir Crit Care    Med, 151, 1915-24.-   34 Kotsimbos, A. T., M. Humbert, et al. (1997). J Allergy Clin    Immunol 99(5): 666-72.-   35 Yamashita N. et al., Cell. Immunol 2002 October; 219(2); 92-97-   36 Ohta K., et al. J Allergy Clin Immunol. 1999 November; 104(5):    1024-30-   37 Stampfli, M. R., R. E. Wiley, et al. (1998). J Clin Invest    102(9): 1704-14.-   38 Cates, E. C., B. U. Gajewska, et al. (2003). J Allergy Clin    Immunol 111(5): 1076-86.-   39 CAMPBELL, I. K., et al. (1997) Annal Res Dis, 56, 364-368.-   40 BISCHOF, R. J., D. ZAFIROPOULOS, J. A. HAMILTON AND I. K.    CAMPBELL (2000) Clin Exp Immunol, 119, 361-367.-   41 Campbell, I. K., M. J. Rich, et al. (1998). J Immunol 161(7):    3639-44.-   42 Hamilton, J. A. (2002). Trends Immunol 23(8): 403-8.-   43 YANG, Y. H. AND J. A. HAMILTON (2001) Arthritis Rheumatol, 44,    111-119-   44 Cook, A. D., E. L. Braine, et al. (2001). Arthritis Res 3(5):    293-8.-   45 Field, M. and L. Clinton (1993). The Lancet 342: 1244.-   46 Firestein G S, Alvaro-Gracia J M, Maki R. 1990 J. Immunol may 1;    144(9): 3347-53-   47 Leizer T, et al. 1990 Blood 1990 Nov. 15; 76(10): 1989-96-   48 Fiehn C, et al., Z Rheumatol 1992 May-June; 51 (3): 1221-126-   49 de Vries, E. G., et al. (1991) Lancet, 338, 517-518.-   50 Hazenberg B P, et al. Blood 1989 December; 74(8): 2769-70-   51 Barnes and Hansel (2004). Lancet, 364:985-96.-   52 Barnes, P. J. (2000). Chest, 117:10 S-14S-   53 McManus, T. E., et al (2005) American Thoracic Society Meeting,    May 2005.-   54 Vlahos, R., et al. (2005). “GM-CSF is a key pathogenic mediator    in experimental COPD.” European Respiratory Society Meeting,    September 2005-   55 McQualter J L, et al. (2001) J Exp Med. 194: 873-82-   56 Bagby G C Jr, et al. 1988 J Clin Invest. 4: 1430-6.-   57 Estrov Z, et al. (1986) Cancer Res. 46: 6456-61.-   58 Barak Y, et al. 1981 Am J Hematol. 10: 269-75.-   59 Gualtieri R J, et al. 1988 Exp Hematol. 16:613-9.-   60 Suda T, et al. 1982 Leuk Res. 6: 43-53.-   61 Gualtieri R J, et al. 1989 Blood. 74: 2360-7-   62 Largaespada D A, et al. (1996) Nat. Genet. 12: 137-43-   63 Iversen, P. O., et al. (1997). Blood 90(12): 4910-7.-   64 Wang et al., 1994 Exp mol Pathol-   65 Plenz et al., Art. Thrombosis and Vascular biology 1997-   66 Takashi et al., 1996 Circulation 93, 1185-1193-   67 Biwa T et al., JBC 1998 273: 28305-28313-   68 Makheja et al., Atherosclerosis 1989, 76 155-661-   69 Naito et al., 1992 J Nutri sci Vitamin 38: 255-264-   70 Hayashi et al., Atherosclerosis 1991 91: 107-116-   71 ViIIa et al 1994 J Clin Invest 93: 1243-   72 Van Put D J et al., 1995 Eur J Pharmacol 294: 753-761-   73 Voisard et al., 1994 Int J Cardiol 43: 257-267-   74 Yamada et al., 1993: Artery 20: 253-267-   75 Sakai et al., 1999 Arterioscler Thromb vasc biol 19: 1726-1733-   76 Gearing, et al. EMBO J. 8 (12): 3667-3676 (1989)-   77 Crosier, K. et al. PNAS 88:7744-7748 1991-   78 Raines, M. et al. PNAS 88:8203-8207 1991-   79 Wess, L. In: BioCentury, The Bernstein Report on BioBusiness,    12(42), A1-A7, 2004-   80 Haan & Maggos (2004) BioCentury, 12(5): A1-A6-   81 Koide et al. (1998) Journal of Molecular Biology, 284: 1141-1151.-   82 Nygren et al. (1997) Current Opinion in Structural Biology, 7:    463-469-   83 Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag    New York, LLC; 2001, ISBN: 3540413545-   84 Mendez, M. et al. (1997) Nature Genet, 15(2): 146-156-   85 Knappik et al. J. Mol. Biol. (2000) 296, 57-86-   86 Krebs et al. Journal of Immunological Methods 254 2001 67-84-   87 Ward, E. S. et al., Nature 341, 544-546 (1989)-   88 McCafferty et al (1990) Nature, 348, 552-554-   89 Holt et al (2003) Trends in Biotechnology 21, 484-490-   90 Bird et al, Science, 242, 423-426, 1988;-   91 Huston et al, PNAS USA, 85, 5879-5883, 1988-   92 Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993-   93 Reiter, Y. et al, Nature Biotech, 14, 1239-1245, 1996-   94 Hu, S. et al, Cancer Res., 56, 3055-3061, 1996.-   95 Holliger, P. and Winter G. Current Opinion Biotechnol 4, 446-449    1993-   96 Ridgeway, J. B. B. et al, Protein Eng., 9, 616-621, 1996-   97 Ann N Y Acad. Sci. 1971 Dec. 31; 190:382-93.-   98 Kabat, E. A. et al, Sequences of Proteins of Immunological    Interest. 4^(th) Edition. US Department of Health and Human    Services. 1987-   99 Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological    Interest, 5th Edition. US Department of Health and Human Services,    Public Service, NIH, Washington.-   100 Persic et al 1997 Gene 187; 9-18-   101 Hanes J et al (2000) Methods in Enzymology, Vol 328:24.-   102 Vaughan T J et al (1996) Nature Biotechnology Vol 14:309-   103 Slootstra-J W; Puijk-W C; Ligtvoet-G J; Langeveld-J P; Meloen-R    H (1996). Mol-Divers. 1: 87-96-   104 WO01/66754 Cambridge Antibody Technology Limited; Vaughan;    Wilton; Smith; Main-   105 P. K. Smith, et al., Anal. Biochem. 150 (1985), pp. 76-85-   106 S. Mizushima and S, Nagata Nucleic Acids Research, Vol 18; No 17    1990 pp 5322-   107 Clin Haematol. 1979 June; 8(2):263-85. Metcalf D.-   108 Nishijima, I., T. Nakahata, et al. (1997). Blood 90(3): 1031-8.

The invention claimed is:
 1. An isolated binding member for humanGM-CSFRα, wherein the binding member binds the extracellular domain ofhuman GM-CSFRα with an affinity (Kd) of 5 nM or less in a surfaceplasmon resonance assay, inhibits binding of GM-CSF to GM-CSFRα, andcomprises a human or humanised antibody molecule that competes forbinding the extracellular domain of human GM-CSFRα with an antibodymolecule having a VH domain amino acid sequence SEQ ID NO: 52 and a VLdomain amino acid sequence SEQ ID NO:
 57. 2. An isolated binding memberfor human GM-CSFRα, wherein the binding member inhibits binding ofGM-CSF to GM-CSFRα and wherein the binding member comprises an antibodymolecule comprising an antibody VH domain comprising a set ofcomplementarity determining regions HCDR1, HCDR2 and HCDR3 and aframework, and an antibody VL domain comprising a set of complementaritydetermining regions LCDR1, LCDR2 and LCDR3 and a framework, wherein theset of complementarity determining regions comprises: SEQ ID NOs: 3, 4,5, 8, 9, and 10; SEQ ID NOs: 3, 4, 15, 8, 9, and 20; SEQ ID NOs: 3, 4,25, 8, 9, and 20; SEQ ID NOs: 3, 4, 35, 8, 9, and 20; SEQ ID NOs: 3, 4,45, 8, 9, and 20; SEQ ID NOs: 3, 4, 55, 8, 9, and 60; SEQ ID NOs: 3, 4,65, 8, 9, and 20; SEQ ID NOs: 3, 4, 75, 8, 9, and 20; SEQ ID NOs: 3, 4,85, 8, 9, and 20; SEQ ID NOs: 3, 4, 95, 8, 9, and 20; SEQ ID NOs: 3, 4,105, 8, 9, and 20; SEQ ID NOs: 3, 4, 115, 8, 9, and 120; SEQ ID NOs: 3,4, 125, 8, 9, and 130; SEQ ID NOs: 3, 4, 135, 8, 9, and 140; SEQ ID NOs:3, 4, 145, 8, 9, and 150; SEQ ID NOs: 3, 4, 155, 8, 9, and 160; SEQ IDNOs: 3, 4, 165, 8, 9, and 170; SEQ ID NOs: 173, 4, 175, 8, 9, and 180;SEQ ID NOs: 3, 4, 185, 8, 9, and 190; or SEQ ID NOs: 3, 4, 195, 8, 9,and
 200. 3. An isolated binding member for human GM-CSFRα, wherein thebinding member comprises an antibody molecule which inhibits binding ofGM-CSF to GM-CSFRα, wherein the binding member binds at least oneresidue of Tyr-Leu-Asp-Phe-Gln at positions 226 to 230 of humanGM-CSFRα, as shown in SEQ ID NO: 206, wherein the binding member bindsto human GM-CSFRα, extra-cellular domain with an affinity (KD) of 5 nMor less in a surface plasmon resonance assay, and wherein the bindingmember comprises an antibody VH domain comprising a set ofcomplementarity determining regions HCDR1, HCDR2 and HCDR3 and aframework, and an antibody VL domain comprising a set of complementaritydetermining regions LCDR1, LCDR2 and LCDR3 and a framework, wherein theset of complementarity determining regions comprises: SEQ ID NOs: 3, 4,5, 8, 9, and 10; SEQ ID NOs: 3, 4, 15, 8, 9, and 20; SEQ ID NOs: 3, 4,25, 8, 9, and 20; SEQ ID NOs: 3, 4, 35, 8, 9, and 20; SEQ ID NOs: 3, 4,45, 8, 9, and 20; SEQ ID NOs: 3, 4, 55, 8, 9, and 60; SEQ ID NOs: 3, 4,65, 8, 9, and 20; SEQ ID NOs: 3, 4, 75, 8, 9, and 20; SEQ ID NOs: 3, 4,85, 8, 9, and 20; SEQ ID NOs: 3, 4, 95, 8, 9, and 20; SEQ ID NOs: 3, 4,105, 8, 9, and 20; SEQ ID NOs: 3, 4, 115, 8, 9, and 120; SEQ ID NOs: 3,4, 125, 8, 9, and 130; SEQ ID NOs: 3, 4, 135, 8, 9, and 140; SEQ ID NOs:3, 4, 145, 8, 9, and 150; SEQ ID NOs: 3, 4, 155, 8, 9, and 160; SEQ IDNOs: 3, 4, 165, 8, 9, and 170; SEQ ID NOs: 173, 4, 175, 8, 9, and 180;SEQ ID NOs: 3, 4, 185, 8, 9, and 190; or SEQ ID NOs: 3, 4, 195, 8, 9,and
 200. 4. An isolated antibody molecule which binds the alpha chain ofhuman Granulocyte/Macrophage Colony Stimulating Factor Receptor(GM-CSFRα) and inhibits binding of GM-CSF to human GM-CSFRα, wherein theantibody molecule comprises: (i) a VH domain comprising a set of VHcomplementarity determining regions (CDRs): CDR1, CDR2 and CDR3 and aframework, wherein the VH domain comprises: (a) VH domain amino acidsequence SEQ ID NO: 52 or (b) a variant of VH domain amino acid sequenceSEQ ID NO: 52 with less than 10 amino acid alterations, and wherein theVH domain comprises: S at Kabat residue H17 of the VH framework; I atKabat residue H34 of VH CDR1; E at Kabat residue H54 of VH CDR2; I atKabat residue H57 of VH CDR2; I, L, V, A or M at Kabat residue H94 ofthe VH framework; V, N, A or L at Kabat residue H95 of VH CDR3; S atKabat residue H97 of VH CDR3; S, F, H, P, T or W at Kabat residue H99 ofVH CDR3; and A, T, P, S, V or H at Kabat residue H100B of VH CDR3; and(ii) a VL domain comprising a set of VL CDRs: CDR1, CDR2 and CDR3 and aframework, wherein the VL domain comprises: (a) VL domain amino acidsequence SEQ ID NO: 57 or 218; or (b) a variant of VL domain amino acidsequence SEQ ID NO: 57 or 218 with less than 10 amino acid alterations,and wherein the VL domain comprises: S at Kabat residue 27A of VL CDR1;N at Kabat residue 27B of VL CDR1; I at Kabat residue 27C of VL CDR1; Dat Kabat residue 32of VL CDR1; N at Kabat residue 51 of VL CDR1; N atKabat residue 52 of VL CDR2; K at Kabat residue 53 of VL CDR2; S, T or Mat Kabat residue L90 of VL CDR3; D, E, Q, S, M or T at Kabat residue L92of VL CDR3; and S, P, I or V at Kabat residue L96 of VL CDR3 providedthat the functionality of the antibody molecule is preserved.
 5. Anisolated antibody molecule comprising a VH domain and a VL domainselected from the following: VH domain SEQ ID NO: 2 and VL domain SEQ IDNO: 208; VH domain SEQ ID NO: 12 and VL domain SEQ ID NO: 210; VH domainSEQ ID NO: 32 and VL domain SEQ ID NO: 210; VH domain SEQ ID NO: 42 andVL domain SEQ ID NO: 210; VH domain SEQ ID NO: 52 and VL domain SEQ IDNO: 218; VH domain SEQ ID NO: 62 and VL domain SEQ ID NO: 210; VH domainSEQ ID NO: 72 and VL domain SEQ ID NO: 210; VH domain SEQ ID NO: 82 andVL domain SEQ ID NO: 210; VH domain SEQ ID NO: 92 and VL domain SEQ IDNO: 210; VH domain SEQ ID NO: 102 and VL domain SEQ ID NO: 228; VHdomain SEQ ID NO: 22 and VL domain SEQ ID NO: 230; VH domain SEQ ID NO:22 and VL domain SEQ ID NO: 232; VH domain SEQ ID NO: 132 and VL domainSEQ ID NO: 234; VH domain SEQ ID NO: 142 and VL domain SEQ ID NO: 236;VH domain SEQ ID NO: 152 and VL domain SEQ ID NO: 238; VH domain SEQ IDNO: 152 and VL domain SEQ ID NO: 240; VH domain SEQ ID NO: 172 and VLdomain SEQ ID NO: 242; VH domain SEQ ID NO: 182 and VL domain SEQ ID NO:244; and VH domain SEQ ID NO: 192 and VL domain SEQ ID NO:
 246. 6. Theisolated binding member of any of claim 1, 2 or 3, or the isolatedantibody molecule of claim 4 or claim 5, wherein the antibody moleculeis a human or humanized antibody molecule.
 7. The antibody molecule ofclaim 6, wherein the human or humanized antibody molecule comprises aframework and wherein the framework comprises: (i) a VH domainframework, wherein the VH domain framework is a human germline VH1 DP5or VH3 DP47 framework, (ii) a VL domain framework, wherein the VL domainframework is a human germline VLambda 1 DPL8, VLambda 1 DPL3 or VLambda6_(—)6a framework; or (iii) both (i) and (ii).
 8. The isolated bindingmember of any of claim 1, 2 or 3, or the isolated antibody molecule ofclaim 4 or claim 5, wherein the antibody molecule is IgG4.
 9. Theisolated binding member of any of claim 1, 2 or 3, or the isolatedantibody molecule of claim 4 or claim 5, which binds human GM-CSFRαextra-cellular domain with an affinity (KD) of 1 nM or less in a surfaceplasmon resonance assay.
 10. A composition comprising the isolatedbinding member of any of claim 1, 2 or 3, or the isolated antibodymolecule of claim 4 or claim 5, and a pharmaceutically acceptableexcipient.
 11. An isolated nucleic acid molecule comprising a nucleicacid sequence encoding the isolated binding member of any of claim 1, 2or 3, or the isolated antibody molecule of claim 4 or claim
 5. 12. Anisolated host cell containing the nucleic acid molecule according toclaim
 11. 13. A method of producing the isolated binding member of anyof claim 1, 2 or 3, or the isolated antibody molecule of claim 4 orclaim 5, comprising culturing host cells containing a nucleic acidmolecule comprising a nucleic acid sequence encoding the binding memberor antibody molecule.
 14. The method according to claim 13, furthercomprising purifying the antibody molecule.
 15. A method of treatingrheumatoid arthritis, in a subject, the method comprising administeringthe isolated binding member of any of claim 1, 2 or 3, or the isolatedantibody molecule of claim 4 or claim 5, to the subject.